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The lab is concerned with mechanisms of cellular immune reactions and tissue injury in the central nervous system (CNS) as they occur in human multiple sclerosis (MS. CNS tissue samples from animals with experimental autoimmune encephalomyelitis (EAE), a model of MS, and from patients with MS and other CNS diseases are studied using histology and immunohistochemistry at the light microscopic and ultrastructural levels to visualize directly the locations of molecules that are involved in immune-mediated injury and the failure of tissue repair.

We are currently focusing on cross-recognition of neurons and neuronal precursors by anti-myelin proteolipid protein antibodies, which may result in neuronal dysfunction and the failure of neuronal repair in CNS injury and on an oligodendrogliopathy induced by the environmental agent azetidine.

Abstract

Aquaporin-4 (AQP4)-specific T cells are expanded in neuromyelitis optica (NMO) patients and exhibit Th17 polarization. However, their pathogenic role in CNS autoimmune inflammatory disease is unclear. Although multiple AQP4 T-cell epitopes have been identified in WT C57BL/6 mice, we observed that neither immunization with those determinants nor transfer of donor T cells targeting them caused CNS autoimmune disease in recipient mice. In contrast, robust proliferation was observed following immunization of AQP4-deficient (AQP4(-/-)) mice with AQP4 peptide (p) 135-153 or p201-220, peptides predicted to contain I-A(b)-restricted T-cell epitopes but not identified in WT mice. In comparison with WT mice, AQP4(-/-) mice used unique T-cell receptor repertoires for recognition of these two AQP4 epitopes. Donor T cells specific for either determinant from AQP4(-/-), but not WT, mice induced paralysis in recipient WT and B-cell-deficient mice. AQP4-specific Th17-polarized cells induced more severe disease than Th1-polarized cells. Clinical signs were associated with opticospinal infiltrates of T cells and monocytes. Fluorescent-labeled donor T cells were detected in CNS lesions. Visual system involvement was evident by changes in optical coherence tomography. Fine mapping of AQP4 p201-220 and p135-153 epitopes identified peptides within p201-220 but not p135-153, which induced clinical disease in 40% of WT mice by direct immunization. Our results provide a foundation to evaluate how AQP4-specific T cells contribute to AQP4-targeted CNS autoimmunity (ATCA) and suggest that pathogenic AQP4-specific T-cell responses are normally restrained by central tolerance, which may be relevant to understanding development of AQP4-reactive T cells in NMO.

Abstract

Over the past few years, MRI has become an indispensable tool for diagnosing multiple sclerosis (MS). However, the current MRI criteria for MS diagnosis have imperfect sensitivity and specificity. The central vein sign (CVS) has recently been proposed as a novel MRI biomarker to improve the accuracy and speed of MS diagnosis. Evidence indicates that the presence of the CVS in individual lesions can accurately differentiate MS from other diseases that mimic this condition. However, the predictive value of the CVS for the development of clinical MS in patients with suspected demyelinating disease is still unknown. Moreover, the lack of standardization for the definition and imaging of the CVS currently limits its clinical implementation and validation. On the basis of a thorough review of the existing literature on the CVS and the consensus opinion of the members of the North American Imaging in Multiple Sclerosis (NAIMS) Cooperative, this article provides statements and recommendations aimed at helping radiologists and neurologists to better understand, refine, standardize and evaluate the CVS in the diagnosis of MS.

Abstract

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-?, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

Abstract

CD48 (SLAMF2) is an adhesion and costimulatory molecule constitutively expressed on hematopoietic cells. Polymorphisms in CD48 have been linked to susceptibility to multiple sclerosis (MS), and altered expression of the structurally related protein CD58 (LFA-3) is associated with disease remission in MS. We examined CD48 expression and function in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We found that a subpopulation of CD4(+) T cells highly upregulated CD48 expression during EAE and were enriched for pathogenic CD4(+) T cells. These CD48(++)CD4(+) T cells were predominantly CD44(+) and Ki67(+), included producers of IL-17A, GM-CSF, and IFN-?, and were most of the CD4(+) T cells in the CNS. Administration of anti-CD48 mAb during EAE attenuated clinical disease, limited accumulation of lymphocytes in the CNS, and reduced the number of pathogenic cytokine-secreting CD4(+) T cells in the spleen at early time points. These therapeutic effects required CD48 expression on CD4(+) T cells but not on APCs. Additionally, the effects of anti-CD48 were partially dependent on Fc?Rs, as anti-CD48 did not ameliorate EAE or reduce the number of cytokine-producing effector CD4(+) T cells in Fc?r1?(-/-) mice or in wild-type mice receiving anti-CD16/CD32 mAb. Our data suggest that anti-CD48 mAb exerts its therapeutic effects by both limiting CD4(+) T cell proliferation and preferentially eliminating pathogenic CD48(++)CD4(+) T cells during EAE. Our findings indicate that high CD48 expression is a feature of pathogenic CD4(+) T cells during EAE and point to CD48 as a potential target for immunotherapy.

Abstract

To investigate the role of very late antigen-4 (VLA-4) on regulatory B cells (Breg) in CNS autoimmune disease.Experimental autoimmune encephalomyelitis (EAE) was induced in mice selectively deficient for VLA-4 on B cells (CD19cre/?4(f/f)) by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p)35-55 or recombinant human (rh) MOG protein. B-cell and T-cell populations were examined by flow cytometry and immunohistochemistry. Breg were evaluated by intracellular IL-10 staining of B cells and, secondly, by coexpression of CD1d and CD5.As previously reported, EAE was less severe in B-cell VLA-4-deficient vs control CD19cre mice when induced by rhMOG, a model that is B-cell-dependent and leads to efficient B-cell activation and antibody production. Paradoxically, B-cell VLA-4-deficient mice developed more severe clinical disease than control mice when EAE was induced with MOG p35-55, a B-cell-independent encephalitogen that does not efficiently activate B cells. Peripheral T-cell and humoral immune responses were not altered in B-cell VLA-4-deficient mice. In MOG p35-55-induced EAE, B-cell VLA-4 deficiency reduced CNS accumulation of B but not T cells. Breg were detected in the CNS of control mice with MOG p35-55-induced EAE. However, more severe EAE in B-cell VLA-4-deficient mice was associated with virtual absence of CNS Breg.Our results demonstrate that CNS accumulation of Breg is VLA-4-dependent and suggest that Breg may contribute to regulation of CNS autoimmunity in situ. These observations underscore the need to choose the appropriate encephalitogen when studying how B cells contribute to pathogenesis or regulation of CNS autoimmunity.

Abstract

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4(+)Foxp3(-) (T conv) and regulatory Foxp3(+) (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.

Abstract

Natalizumab, which binds very late antigen-4 (VLA-4), is a potent therapy for multiple sclerosis (MS). Studies have focused primarily upon its capacity to interfere with T-cell migration into the central nervous system (CNS). B cells are important in MS pathogenesis and express high levels of VLA-4. Here, we report that the selective inhibition of VLA-4 expression on B cells impedes CNS accumulation of B cells, and recruitment of Th17 cells and macrophages, and reduces susceptibility to experimental autoimmune encephalomyelitis. These results underscore the importance of B-cell VLA-4 expression in the pathogenesis of CNS autoimmunity and provide insight regarding mechanisms that may contribute to the benefit of natalizumab in MS, as well as candidate therapeutics that selectively target B cells.

Abstract

T cell Ig and mucin domain (Tim)-1 identifies IL-10-producing regulatory B cells (Bregs). Mice on the C57BL/6 background harboring a loss-of-function Tim-1 mutant showed progressive loss of IL-10 production in B cells and with age developed severe multiorgan tissue inflammation. We demonstrate that Tim-1 expression and signaling in Bregs are required for optimal production of IL-10. B cells with Tim-1 defects have impaired IL-10 production but increased proinflammatory cytokine production, including IL-1 and IL-6. Tim-1-deficient B cells promote Th1 and Th17 responses but inhibit the generation of regulatory T cells (Foxp3(+) and IL-10-producing type 1 regulatory T cells) and enhance the severity of experimental autoimmune encephalomyelitis. Mechanistically, Tim-1 on Bregs is required for apoptotic cell (AC) binding to Bregs and for AC-induced IL-10 production in Bregs. Treatment with ACs reduces the severity of experimental autoimmune encephalomyelitis in hosts with wild-type but not Tim-1-deficient Bregs. Collectively, these findings suggest that in addition to serving as a marker for identifying IL-10-producing Bregs, Tim-1 is also critical for maintaining self-tolerance by regulating IL-10 production in Bregs.

Abstract

Podoplanin (PDPN, also known as Gp38) is highly expressed on the surface of lymphatic endothelial cells, where it regulates development of lymphatic vessels. We have recently observed that PDPN is also expressed on effector T cells that infiltrate target tissues during autoimmune inflammation; however, the function of PDPN in T cells is largely unclear. Here, we demonstrated that global deletion of Pdpn results in exaggerated T cell responses and spontaneous experimental autoimmune encephalomyelitis (EAE) in mice with a susceptible genetic background. In contrast, T cell-specific overexpression of PDPN resulted in profound defects in IL-7-mediated T cell expansion and survival. Consequently, these animals exhibited a more rapid resolution of CNS inflammation, characterized by a reduced effector CD4+ T cell population in the CNS. Mice harboring a T cell-specific deletion of Pdpn developed exacerbated EAE, with increased accumulation of effector CD4+ T cells in the CNS. Transcriptional profiling of naturally occurring PDPN+ effector T cells in the CNS revealed increased expression of other inhibitory receptors, such as Pd1 and Tim3, and decreased expression of prosurvival factors, including Il7ra. Together, our data suggest that PDPN functions as an inhibitory molecule on T cells, thereby promoting tissue tolerance by limiting long-term survival and maintenance of CD4+ effector T cells in target organs.

Abstract

We recently reported that Acanthamoeba castellanii (ACA), an opportunistic pathogen of the central nervous system (CNS) possesses mimicry epitopes for proteolipid protein (PLP) 139-151 and myelin basic protein 89-101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE) in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139-151, as evaluated by T cell proliferation and IAs/dextramer staining. We verified that PLP 139-151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokine-producing cells, and they can transfer disease to naïve animals. Likewise, the animals first primed with suboptimal dose of PLP 139-151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis.

Abstract

Using an integrated antigen microarray approach, we observed epitope-spreading of autoantibody responses to a variety of antigenic structures in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and in the serum of mice with experimental autoimmune encephalomyelitis (EAE). These included previously described protein- and lipid-based antigenic targets and newly discovered autoimmunogenic sugar moieties, notably, autoantibodies specific for the oligomannoses in both MS patient CSF and the sera of mice with EAE. These glycans are often masked by other sugar moieties and belong to a class of cryptic autoantigens. We further determined that these targets are highly expressed on multiple cell types in MS and EAE lesions. Co-immunization of SJL/J mice with a Man9-KLH conjugate at the time of EAE induction elicited highly significant levels of anti-Man9-cluster autoantibodies. Nevertheless, this anti-glycan autoantibody response was associated with a significantly reduced clinical severity of EAE. The potential of these cryptic glycan markers and targeting antibodies for diagnostic and therapeutic interventions of neurological disorders has yet to be explored.

Abstract

Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(?-naphthoyl) ethyltrimethylammonium iodide (?-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated ?-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. ?-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, ?-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.

Abstract

Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould's angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections.

Abstract

The amyloid-forming proteins tau, ?B crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein ?B crystallin (HspB5) and from amyloid ? fibrils, characteristic of Alzheimer's disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, we showed a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid ? A4, major prion protein (PrP), HspB5, amylin, serum amyloid P, and insulin B chain, to be anti-inflammatory and capable of reducing serological levels of interleukin-6 and attenuating paralysis in EAE. The chaperone function of the fibrils correlates with the therapeutic outcome. Fibrils composed of tau 623-628 precipitated 49 plasma proteins, including apolipoprotein B-100, clusterin, transthyretin, and complement C3, supporting the hypothesis that the fibrils are active biological agents. Amyloid fibrils thus may provide benefit in MS and other neuroinflammatory disorders.

Abstract

To determine whether the therapeutic activity of ?B crystallin, small heat shock protein B5 (HspB5), was shared with other human sHsps, a set of seven human family members, a mutant of HspB5 G120 known to exhibit reduced chaperone activity, and a mycobacterial sHsp were expressed and purified from bacteria. Each of the recombinant proteins was shown to be a functional chaperone, capable of inhibiting aggregation of denatured insulin with varying efficiency. When injected into mice at the peak of disease, they were all effective in reducing the paralysis in experimental autoimmune encephalomyelitis. Additional structure activity correlations between chaperone activity and therapeutic function were established when linear regions within HspB5 were examined. A single region, corresponding to residues 73-92 of HspB5, forms amyloid fibrils, exhibited chaperone activity, and was an effective therapeutic for encephalomyelitis. The linkage of the three activities was further established by demonstrating individual substitutions of critical hydrophobic amino acids in the peptide resulted in the loss of all of the functions.

Abstract

Interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are often present at the sites of tissue inflammation in autoimmune diseases, which has led to the conclusion that T(H)17 cells are main drivers of autoimmune tissue injury. However, not all T(H)17 cells are pathogenic; in fact, T(H)17 cells generated with transforming growth factor-?1 (TGF-?1) and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we found that the production of TGF-?3 by developing T(H)17 cells was dependent on IL-23, which together with IL-6 induced very pathogenic T(H)17 cells. Moreover, TGF-?3-induced T(H)17 cells were functionally and molecularly distinct from TGF-?1-induced T(H)17 cells and had a molecular signature that defined pathogenic effector T(H)17 cells in autoimmune disease.

Abstract

Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1-mutant mouse (Tim-1(?mucin)) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1(?mucin) mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-? and elevated serum levels of Ig and autoantibodies. However, Tim-1(?mucin) mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1(?mucin)lpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.

Abstract

Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47(-/-) mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein ? (SIRP-?). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.

Abstract

Laquinimod is a novel oral drug that is currently being evaluated for the treatment of relapsing-remitting (RR) multiple sclerosis (MS). Using the animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we examined how laquinimod promotes immune modulation. Oral laquinimod treatment reversed established RR-EAE and was associated with reduced central nervous system (CNS) inflammation, decreased Th1 and Th17 responses, and an increase in regulatory T cells (Treg). In vivo laquinimod treatment inhibited donor myelin-specific T cells from transferring EAE to naive recipient mice. In vivo laquinimod treatment altered subpopulations of myeloid antigen presenting cells (APC) that included a decrease in CD11c(+)CD11b(+)CD4(+) dendritic cells (DC) and an elevation of CD11b(hi)Gr1(hi) monocytes. CD11b(+) cells from these mice exhibited an anti-inflammatory type II phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, donor type II monocytes from laquinimod-treated mice suppressed clinical and histologic disease in recipients with established EAE. As effects were observed in both APC and T cell compartments, we examined whether T cell immune modulation occurred as a direct effect of laquinimod on T cells, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes or DC from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Thus, laquinimod modulates adaptive T cell immune responses via its effects on cells of the innate immune system, and may not influence T cells directly.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the ?- and ?-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.

Abstract

Analyses of varicella-zoster virus (VZV) protein expression during latency have been discordant, with rare to many positive neurons detected. We show that ascites-derived murine and rabbit antibodies specific for VZV proteins in vitro contain endogenous antibodies that react with human blood type A antigens in neurons. Apparent VZV neuronal staining and blood type A were strongly associated (by a ?² test, ? = 0.0003). Adsorption of ascites-derived monoclonal antibodies or antiserum with type A erythrocytes or the use of in vitro-derived VZV monoclonal antibodies eliminated apparent VZV staining. Animal-derived antibodies must be screened for anti-blood type A reactivity to avoid misidentification of viral proteins in the neurons of the 30 to 40% of individuals who are blood type A.

Abstract

Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögren's syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.

Abstract

Multiple sclerosis is an autoimmune disease of the central nervous system characterized by neuroinflammation and demyelination. Although considered a T cell-mediated disease, multiple sclerosis involves the activation of both adaptive and innate immune cells, as well as resident cells of the central nervous system, which synergize in inducing inflammation and thereby demyelination. Differentiation, survival, and inflammatory functions of innate immune cells and of astrocytes of the central nervous system are regulated by tyrosine kinases. Here, we show that imatinib, sorafenib, and GW2580-small molecule tyrosine kinase inhibitors-can each prevent the development of disease and treat established disease in a mouse model of multiple sclerosis. In vitro, imatinib and sorafenib inhibited astrocyte proliferation mediated by the tyrosine kinase platelet-derived growth factor receptor (PDGFR), whereas GW2580 and sorafenib inhibited macrophage tumor necrosis factor (TNF) production mediated by the tyrosine kinases c-Fms and PDGFR, respectively. In vivo, amelioration of disease by GW2580 was associated with a reduction in the proportion of macrophages and T cells in the CNS infiltrate, as well as a reduction in the levels of circulating TNF. Our findings suggest that GW2580 and the FDA-approved drugs imatinib and sorafenib have potential as novel therapeutics for the treatment of autoimmune demyelinating disease.

Abstract

Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase C? (PKC?) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKC? binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKC? is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKC?, using a selective PKC? peptide inhibitor (?V1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKC? activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.

Abstract

Peroxisome proliferator-activated receptors (PPARs; PPAR-alpha, PPAR-delta, and PPAR-gamma) comprise a family of nuclear receptors that sense fatty acid levels and translate this information into altered gene transcription. Previously, it was reported that treatment of mice with a synthetic ligand activator of PPAR-delta, GW0742, ameliorates experimental autoimmune encephalomyelitis (EAE), indicating a possible role for this nuclear receptor in the control of central nervous system (CNS) autoimmune inflammation. We show that mice deficient in PPAR-delta (PPAR-delta(-/-)) develop a severe inflammatory response during EAE characterized by a striking accumulation of IFN-gamma(+)IL-17A(-) and IFN-gamma(+)IL-17A(+) CD4(+) cells in the spinal cord. The preferential expansion of these T helper subsets in the CNS of PPAR-delta(-/-) mice occurred as a result of a constellation of immune system aberrations that included higher CD4(+) cell proliferation, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. We also show that the effect of PPAR-delta in inhibiting the production of IFN-gamma and IL-12 family cytokines is ligand dependent and is observed in both mouse and human immune cells. Collectively, these findings suggest that PPAR-delta serves as an important molecular brake for the control of autoimmune inflammation.

Abstract

IFN-gamma plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-gamma-producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Molecular characterization of CD11b(+)Ly-6G(+) cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b(+)Ly-6G(+) cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

Abstract

Mn superoxide dismutase (MnSOD) is an important mitochondrial antioxidant enzyme, and elevated MnSOD levels have been shown to reduce tumor growth in part by suppressing cell proliferation. Studies with fibroblasts have shown that increased MnSOD expression prolongs cell cycle transition time in G1/S and favors entrance into the quiescent state. To determine if the same effect occurs during tissue regeneration in vivo, we used a transgenic mouse system with liver-specific MnSOD expression and a partial hepatectomy paradigm to induce synchronized in vivo cell proliferation during liver regeneration. We show in this experimental system that a 2.6-fold increase in MnSOD activity leads to delayed entry into S phase, as measured by reduction in bromodeoxyuridine (BrdU) incorporation and decreased expression of proliferative cell nuclear antigen (PCNA). Thus, compared to control mice with baseline MnSOD levels, transgenic mice with increased MnSOD expression in the liver have 23% fewer BrdU-positive cells and a marked attenuation of PCNA expression. The increase in MnSOD activity also leads to an increase in the mitochondrial form of thioredoxin (thioredoxin 2), but not in several other peroxidases examined, suggesting the importance of thioredoxin 2 in maintaining redox balance in mitochondria with elevated levels of MnSOD.

Abstract

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a model of human multiple sclerosis induced by autoreactive Th cells that mediate tissue inflammation and demyelination in the CNS. Initially, IFN-gamma-producing Th1 cells and, more recently, IL-17-producing Th17 cells with specificity for myelin Ags have been implicated in EAE induction, but whether Th17 cells are encephalitogenic has been controversial. Moreover, a new effector T cell subset, Th9 cells, has been identified; however, the ability of this T cell subset to induce EAE has not been investigated. Here, we have developed protocols to generate myelin oligodendrocyte glycoprotein-specific Th17, Th1, Th2, and Th9 cells in vitro, so that we could directly compare and characterize the encephalitogenic activity of each of these subsets upon adoptive transfer. We show that myelin oligodendrocyte glycoprotein-specific Th1, Th17, and Th9 cells but not Th2 cells induce EAE upon adoptive transfer. Importantly, each T cell subset induced disease with a different pathological phenotype. These data demonstrate that different effector T cell subsets with specificity for myelin Ags can induce CNS autoimmunity and that the pathological heterogeneity in multiple sclerosis lesions might in part be due to multiple distinct myelin-reactive effector T cells.

Abstract

We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35-55 (MOG(35-55)), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG(35-55)-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.

Abstract

Idd5.1 regulates T1D susceptibility in nonobese diabetic (NOD) mice and has two notable candidate genes, Ctla4 and Icos. Reduced expression of one of the four CTLA-4 isoforms, ligand-independent CTLA-4 (liCTLA-4), which inhibits in vitro T cell activation and cytokine production similarly to full-length CTLA-4 (flCTLA-4), has been hypothesized to increase type 1 diabetes (T1D) susceptibility. However, further support of this hypothesis is required since the Idd5.1 haplotypes of the diabetes-susceptible NOD and the resistant B10 strains differ throughout Ctla4 and Icos. Using haplotype analysis and the generation of novel Idd5.1-congenic strains that differ at the disease-associated Ctla4 exon 2 single-nucleotide polymorphism, we demonstrate that increased expression of liCTLA-4 correlates with reduced T1D susceptibility. To directly assess the ability of liCTLA-4 to modulate T1D, we generated liCTLA-4-transgenic NOD mice and compared their diabetes susceptibility to nontransgenic littermates. NOD liCTLA-4-transgenic mice were protected from T1D to the same extent as NOD.B10 Idd5.1-congenic mice, demonstrating that increased liCTLA-4 expression alone can account for disease protection. To further investigate the in vivo function of liCTLA-4, specifically whether liCTLA-4 can functionally replace flCTLA-4 in vivo, we expressed the liCTLA-4 transgene in CTLA-4(-/-) B6 mice. CTLA-4(-/-) mice expressing liCTLA-4 accumulated fewer activated effector/memory CD4(+) T cells than CTLA-4(-/-) mice and the transgenic mice were partially rescued from the multiorgan inflammation and early lethality caused by the disruption of Ctla4. These results suggest that liCTLA-4 can partially replace some functions of flCTLA-4 in vivo and that this isoform evolved to reinforce the function of flCTLA-4.

Abstract

The renin-angiotensin-aldosterone system (RAAS) is a major regulator of blood pressure. The octapeptide angiotensin II (AII) is proteolytically processed from the decapeptide AI by angiotensin-converting enzyme (ACE), and then acts via angiotensin type 1 and type 2 receptors (AT1R and AT2R). Inhibitors of ACE and antagonists of the AT1R are used in the treatment of hypertension, myocardial infarction, and stroke. We now show that the RAAS also plays a major role in autoimmunity, exemplified by multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Using proteomics, we observed that RAAS is up-regulated in brain lesions of MS. AT1R was induced in myelin-specific CD4+ T cells and monocytes during autoimmune neuroinflammation. Blocking AII production with ACE inhibitors or inhibiting AII signaling with AT1R blockers suppressed autoreactive TH1 and TH17 cells and promoted antigen-specific CD4+FoxP3+ regulatory T cells (Treg cells) with inhibition of the canonical NF-kappaB1 transcription factor complex and activation of the alternative NF-kappaB2 pathway. Treatment with ACE inhibitors induces abundant CD4+FoxP3+ T cells with sufficient potency to reverse paralytic EAE. Modulation of the RAAS with inexpensive, safe pharmaceuticals used by millions worldwide is an attractive therapeutic strategy for application to human autoimmune diseases.

Abstract

In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 x 10(4) Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBi-treated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations.

Abstract

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.

Abstract

Voltage-gated Ca(2+) channels (VGCCs) are membrane proteins that determine the activity and survival of neurons, and mutations in the P/Q-type VGCCs are known to cause cerebellar ataxia. VGCC dysfunction may also underlie acquired peripheral and central nervous system diseases associated with small-cell lung cancer, including Lambert-Eaton myasthenic syndrome (LEMS) and paraneoplastic cerebellar ataxia (PCA). The pathogenic role of anti-VGCC antibody in LEMS is well established. Although anti-VGCC antibody is also found in a significant fraction of PCA patients, its contribution to PCA is unclear. Using a polyclonal peptide antibody against a major immunogenic region in P/Q-type VGCCs (the extracellular Domain-III S5-S6 loop), we demonstrated that such antibody was sufficient to inhibit VGCC function in neuronal and recombinant VGCCs, alter cerebellar synaptic transmission, and confer the phenotype of cerebellar ataxia. Our data support the hypothesis that anti-VGCC antibody may play a significant role in the pathogenesis of cerebellar dysfunction in PCA.

Abstract

Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of deltaPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using deltaV1-1 - a selective peptide inhibitor of deltaPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or deltaV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the deltaV1-1-treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with deltaV1-1 was also associated with decreased deltaPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with deltaV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that deltaPKC may specifically act to dysregulate tight junction components. Together, these results suggest that deltaPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.

Abstract

alphaB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease.

Abstract

It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti-Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-gamma and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti-Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-gamma and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti-Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged.

Abstract

Treatment with ex vivo-generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)(35-55)/IA(b) (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer-reactive, Foxp3(+) T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3(+) T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4(+)Foxp3(+) T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.

Abstract

Coccidioidal meningitis (CM) is a devastating disease that requires long-term therapy and for which there is little hope of a cure. A model was used to compare the efficacies of itraconazole and fluconazole. CD-1 mice were infected intrathecally with 30 to 36 viable arthroconidia of Coccidioides. Oral therapy with cyclodextrin (control) or itraconazole or fluconazole at 10, 25, or 50 mg/kg of body weight twice daily (BID) was given for 12 days, from day 3 of infection. Treatment with both antifungals at all doses prolonged survival compared with that of the control treatment (P < 0.01 to 0.0001). At 50 mg/kg, itraconazole and fluconazole were equivalent, whereas itraconazole at 10 or 25 mg/kg prolonged survival compared to that achieved with fluconazole at these dosages (P < 0.05 and 0.01, respectively). Early histologic analysis (10 days of treatment) with 50 mg/kg BID itraconazole or fluconazole showed suppression of CM in all five animals per group; in quantitative cultures, three of three animals from each group had no detectable infection in the brain, spinal cord, or a site of secondary infection, the lungs. In contrast, four of seven controls showed mild to severe meningitis, with arteritis detected in three animals. In a short-term organ clearance study, 5 days of treatment with 10 or 50 mg/kg BID itraconazole or fluconazole reduced the tissue burdens in the brain and spinal cord compared to the tissue burdens in the controls (P < 0.02 to 0.0003). Fluconazole at 10 mg/kg did not reduce the fungal burden in secondary sites, the lungs and kidneys, whereas this itraconazole dose was more effective in clearing the fungi from both organs (P < 0.05 and P < 0.001, respectively). At 50 mg/kg, itraconazole and fluconazole were equivalent in clearing the fungi from the brain and kidney, but itraconazole was superior to fluconazole in clearing the fungi from the spinal cord and lungs (P < 0.05). Thus, both itraconazole and fluconazole were effective at controlling CM, but neither eliminated Coccidioides from tissues. Overall, itraconazole was more efficacious on an mg/kg basis; at high doses they were similarly effective.

Abstract

Peroxisome proliferator-activated receptor (PPAR)alpha is a nuclear receptor that mediates gender differences in lipid metabolism. PPARalpha also functions to control inflammatory responses by repressing the activity of nuclear factor kappaB (NF-kappaB) and c-jun in immune cells. Because PPARalpha is situated at the crossroads of gender and immune regulation, we hypothesized that this gene may mediate sex differences in the development of T cell-mediated autoimmune disease. We show that PPARalpha is more abundant in male as compared with female CD4(+) cells and that its expression is sensitive to androgen levels. Genetic ablation of this gene selectively removed the brake on NF-kappaB and c-jun activity in male T lymphocytes, resulting in higher production of interferon gamma and tumor necrosis factor (but not interleukin 17), and lower production of T helper (Th)2 cytokines. Upon induction of experimental autoimmune encephalomyelitis, male but not female PPARalpha(-/-) mice developed more severe clinical signs that were restricted to the acute phase of disease. These results suggest that males are less prone to develop Th1-mediated autoimmunity because they have higher T cell expression of PPARalpha.

Abstract

Heme oxygenase-1 (HO-1, encoded by HMOX1) dampens inflammatory reactions via the catabolism of heme into CO, Fe, and biliverdin. We report that expression of HO-1 dictates the pathologic outcome of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Induction of EAE in Hmox1(-/- )C57BL/6 mice led to enhanced CNS demyelination, paralysis, and mortality, as compared with Hmox1(+/+) mice. Induction of HO-1 by cobalt protoporphyrin IX (CoPPIX) administration after EAE onset reversed paralysis in C57BL/6 and SJL/J mice and disease relapse in SJL/J mice. These effects were not observed using zinc protoporphyrin IX, which does not induce HO-1. CoPPIX protection was abrogated in Hmox1(-/-) C57BL/6 mice, indicating that CoPPIX acts via HO-1 to suppress EAE progression. The protective effect of HO-1 was associated with inhibition of MHC class II expression by APCs and inhibition of Th and CD8 T cell accumulation, proliferation, and effector function within the CNS. Exogenous CO mimicked these effects, suggesting that CO contributes to the protective action of HO-1. In conclusion, HO-1 or exposure to its end product CO counters autoimmune neuroinflammation and thus might be used therapeutically to treat MS.

Abstract

Relapses and disease exacerbations are vexing features of multiple sclerosis. Osteopontin (Opn), which is expressed in multiple sclerosis lesions, is increased in patients' plasma during relapses. Here, in models of multiple sclerosis including relapsing, progressive and multifocal experimental autoimmune encephalomyelitis (EAE), Opn triggered recurrent relapses, promoted worsening paralysis and induced neurological deficits, including optic neuritis. Increased inflammation followed Opn administration, whereas its absence resulted in more cell death of brain-infiltrating lymphocytes. Opn promoted the survival of activated T cells by inhibiting the transcription factor Foxo3a, by activating the transcription factor NF-kappaB through induction of phosphorylation of the kinase IKKbeta and by altering expression of the proapoptotic proteins Bim, Bak and Bax. Those mechanisms collectively suppressed the death of myelin-reactive T cells, linking Opn to the relapses and insidious progression characterizing multiple sclerosis.

Abstract

Cryptococcus neoformans (Cn) var. grubii or Cryptococcus neoformans var. neoformans infection is usually associated with immunocompromised hosts, whereas Cryptococcusgattii more frequently causes disease in immunocompetent hosts. We examined the effects of immunodeficiency and glucocorticoid-induced immunosuppression on systemic murine infection induced by i.v. inoculation with these pathogens. SCID and immunocompetent BALB/c and C57BL/6 mice were infected with

Abstract

Multiple sclerosis (MS) is a clinically and pathologically heterogeneous inflammatory/demyelinating disease of the CNS. In the MS variant Devic disease, lesions are predominantly found in the optic nerves and spinal cord but not the brain. The immunological bases of the different forms of MS are unknown. We previously generated myelin oligodendrocyte glycoprotein-specific (MOG-specific) TCR transgenic mice (TCRMOG mice; also referred to as 2D2 mice) and reported that a large proportion of these mice develop spontaneous isolated optic neuritis. We have now crossed the TCRMOG mice with MOG-specific Ig heavy-chain knock-in mice (IgHMOG mice; also referred to as Th mice), in which one-third of the B cells are specific for MOG. In these mice, MOG-specific B cells are very efficient in presenting MOG to the transgenic T cells and undergo class switching to IgG1 in the presence of the transgenic T cells. Sixty percent of TCRMOG x IgHMOG mice spontaneously developed a severe form of experimental autoimmune encephalomyelitis (EAE). Histological examination of the CNS revealed a selective distribution of meningeal and parenchymal inflammatory lesions in the spinal cord and optic nerves. Thus, CNS antigen-specific T and B cells cooperate to induce a distinct clinicopathologic EAE pattern that closely replicates human Devic disease.

Abstract

One approach to improving efficacy in MS therapy is to identify medications that provide additive or synergistic benefit in combination. Orally administered cholesterol-lowering HMG-CoA reductase inhibitors (known as statins), which exhibit immunomodulatory properties and are effective in treatment of the MS model EAE, are being tested in MS. As atorvastatin can enhance protective Th2 responses and has a different mechanism of action than glatiramer acetate (GA), a parenterally administered immunomodulatory agent approved for MS treatment, we tested whether the combination of these agents could be beneficial in EAE. Combination therapy using suboptimal doses of atorvastatin and GA prevented or reversed clinical and histologic EAE. Secretion of proinflammatory Th1 cytokines was reduced--and conversely Th2 cytokine secretion was increased--in these mice, but not in mice treated with each drug alone at the same doses. Monocytes treated with the combination of suboptimal doses of atorvastatin and GA secreted an antiinflammatory type II cytokine pattern and, when used as APCs, promoted Th2 differentiation of naive myelin-specific T cells. Our results demonstrate that agents with different mechanisms of immune modulation can combine in a synergistic manner for the treatment of CNS autoimmunity and provide rationale for testing the combination of atorvastatin and GA in MS.

Abstract

Myelin proteolipid protein (PLP), the major protein of mammalian CNS myelin, is a member of the proteolipid gene family (pgf). It is an evolutionarily conserved polytopic integral membrane protein and a potential autoantigen in multiple sclerosis (MS). To analyze antibody recognition of PLP epitopes in situ, monoclonal antibodies (mAbs) specific for different regions of human PLP (50-69, 100-123, 139-151, 178-191, 200-219, 264-276) were generated and used to immunostain CNS tissues of representative vertebrates. mAbs to each region recognized whole human PLP on Western blots; the anti-100-123 mAb did not recognize DM-20, the PLP isoform that lacks residues 116-150. All of the mAbs stained fixed, permeabilized oligodendrocytes and mammalian and avian CNS tissue myelin. Most of the mAbs also stained amphibian, teleost, and elasmobranch CNS myelin despite greater diversity of their pgf myelin protein sequences. Myelin staining was observed when there was at least 40% identity of the mAb epitope and known pgf myelin proteins of the same or related species. The pgf myelin proteins of teleosts and elasmobranchs lack 116-150; the anti-100-123 mAb did not stain their myelin. In addition to myelin, the anti-178-191 mAb stained many neurons in all species; other mAbs stained distinct neuron subpopulations in different species. Neuronal staining was observed when there was at least approximately 30% identity of the PLP mAb epitope and known pgf neuronal proteins of the same or related species. Thus, anti-human PLP epitope mAbs simultaneously recognize CNS myelin and neurons even without extensive sequence identity. Widespread anti-PLP mAb recognition of neurons suggests a novel potential pathophysiologic mechanism in MS patients, i.e., that anti-PLP antibodies associated with demyelination might simultaneously recognize pgf epitopes in neurons, thereby affecting their functions.

Abstract

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.

Abstract

Local catabolism of the amino acid tryptophan (Trp) by indoleamine 2,3-dioxygenase (IDO) is considered an important mechanism of regulating T cell immunity. We show that IDO transcription was increased when myelin-specific T cells were stimulated with tolerogenic altered self-peptides. Catabolites of Trp suppressed proliferation of myelin-specific T cells and inhibited production of proinflammatory T helper-1 (T(H)1) cytokines. N-(3,4,-Dimethoxycinnamoyl) anthranilic acid (3,4-DAA), an orally active synthetic derivative of the Trp metabolite anthranilic acid, reversed paralysis in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis (MS). Trp catabolites and their derivatives offer a new strategy for treating T(H)1-mediated autoimmune diseases such as MS.

Abstract

Immunoglobulin A (IgA), the predominant immunoglobulin class in mucosal secretions, has been found in the cerebrospinal fluid of patients with multiple sclerosis (MS). In this study we examined the infiltration of clonally expanded IgA plasma cells in lesions of MS brains. Sequences of complementarity-determining region 3 of IgA variable heavy chain (V(H)) genes demonstrated the clonal expansion of IgA-bearing plasma cells in MS lesions. Somatic mutations and ongoing intra-clonal mutations occurred in their V(H) genes. Immunohistochemical study demonstrated infiltration of dimer and polymer IgA1- and A2-positive plasma cells in perivascular spaces, in the parenchyma of MS lesions, and in the adjacent white matter. Double immunofluorescence staining showed binding of IgA antibody on axons and walls of microvessels in the areas of chronic active and inactive demyelination. Bielshowsky's silver impregnation revealed axonal damage in these areas. These findings suggest that IgA in the CNS are localized on axons in lesions and may contribute to axonal damage in MS.

Abstract

Surface molecules that are differentially expressed on Th1 and Th2 cells may be useful in regulating specific immune responses in vivo. Using a panel of mAbs, we have identified murine CD226 as specifically expressed on the surface of differentiated Th1 cells but not Th2 or Th0 cells. Although CD226 is constitutively expressed on CD8 cells, it is up-regulated on CD4 cells upon activation. Th1 differentiation results in enhanced CD226 expression, whereas expression is down-regulated upon Th2 polarization. We demonstrate that CD226 is involved in the regulation of T cell activation; in vivo treatment with anti-CD226 results in significant reduction of Th1 cell expansion and in the induction of APCs that inhibit T cell activation. Furthermore, anti-CD226 treatment delays the onset and reduces the severity of a Th1-mediated autoimmune disease, experimental autoimmune encephalomyelitis. Our data suggest that CD226 is a costimulatory molecule that plays an important role in activation and effector functions of Th1 cells.

Abstract

Identification of the T cell immunoglobulin mucin-domain containing (Tim) gene family introduced a new family of cell surface molecules that is involved in the regulation of immune responses. We previously demonstrated that Tim-3 is expressed on terminally differentiated T helper (Th)1 cells, and serves to regulate Th1 immune responses. Here, we describe the identification and function of Tim-2, a novel member of the Tim gene family. In contrast with Tim-3, we demonstrate that Tim-2 is expressed preferentially in differentiated Th2 cells. Blockade of the Tim-2/Tim-2 ligand interaction, by administration of soluble Tim-2 fusion protein (Tim-2 immunoglobulin [Ig]), results in T cell hyperproliferation and the production of Th2 cytokines. Administration of Tim-2 Ig during the induction phase reduces the severity of experimental autoimmune encephalomyelitis, a Th1-mediated autoimmune disease model of multiple sclerosis. We propose that Tim-2, an orthologue of human Tim-1, is critical for the regulation of Th2 responses during autoimmune inflammation.

Abstract

Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.

Abstract

Complexes of the tyrosine kinase ephrin ligands (ephrins) and their receptors (Ephs) provide critical cell recognition signals in CNS development. Complementary ephrin/Eph expression gradients present topographic guidance cues that may either stimulate or repulse axon growth. Some ephrin/Ephs are upregulated in adult CNS injury models. To assess their involvement in multiple sclerosis (MS), ephrin A1-5 and Eph A1-8 expression was analyzed in CNS tissues using immunohistochemistry. Control samples showed distinct expression patterns for each ephrin/Eph on different cell types. Perivascular mononuclear inflammatory cells, reactive astrocytes and macrophages expressed ephrin A1-4, Eph A1, -A3, -A4, -A6 and -A7 in active MS lesions. Axonal ephrin A1 and Eph A3, -A4, and -A7 expression was increased in active lesions and was greater in normal-appearing white matter (NAWM) adjacent to active lesions than within or adjacent to chronic MS lesions, in contralateral NAWM, or in control samples. As in development, therefore, there are temporally dynamic, lesion-associated axonal ephrin/Eph A expression gradients in the CNS of MS patients. These results indicate that ephrin/Eph As are useful cell markers in human CNS tissue samples; they likely are involved in the immunopathogenesis of active lesions and in neurodegeneration in MS NAWM; and they represent potential therapeutic targets in MS.

Abstract

Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has been proposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery in animal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterize the immune response to Nogo-66 and to determine the role of Nogo in experimental autoimmune encephalomyelitis (EAE). Immunization of EAE-susceptible mouse strains with peptides derived from Nogo-66 induced a CNS immune response with clinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive to other encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a wide spectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads to several other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, via Nogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66 are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic.

Abstract

SJL mice are highly susceptible to experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, whereas H-2 congenic B10.S mice are resistant. Immunodominance and susceptibility to EAE are associated with a high precursor frequency of PLP 139-151-specific T cells in the naive repertoire of SJL mice. To understand the mechanism of EAE resistance in B10.S mice, we determined the precursor frequency of PLP 139-151-reactive T cells in both strains by using IAs/PLP 139-151 tetramers. SJL and B10.S mice had similar frequencies of tetramer-reactive T cells in the naive peripheral repertoire. However, in SJL mice, the majority of PLP 139-151 tetramer-positive cells were in the CD4+CD25- population, whereas there were more tetramer-positive cells in the CD4+CD25+ population of B10.S mice. Depletion of CD4+CD25+ cells in vivo facilitated the expansion of PLP 139-151-reactive cells with production of T helper 1 cytokines in EAE-resistant B10.S mice. Furthermore, anti-CD25 Ab treatment before immunization resulted in EAE induction in these otherwise resistant mice. These data indicate an important role for autoantigen-specific CD4+CD25+ cells in genetic resistance to autoimmunity.

Abstract

Both positive and negative regulatory roles have been suggested for the B7 family member PD-L1(B7-H1). PD-L1 is expressed on antigen-presenting cells (APCs), activated T cells, and a variety of tissues, but the functional significance of PD-L1 on each cell type is not yet clear. To dissect the functions of PD-L1 in vivo, we generated PD-L1-deficient (PD-L1(-/-)) mice. CD4(+) and CD8(+) T cell responses were markedly enhanced in PD-L1(-/-) mice compared with wild-type mice in vitro and in vivo. PD-L1(-/-) dendritic cells stimulated greater wild-type CD4(+) T cell responses than wild-type dendritic cells, and PD-L1(-/-) CD4(+) T cells produced more cytokines than wild-type CD4(+) T cells in vitro, demonstrating an inhibitory role for PD-L1 on APCs and T cells. In vivo CD8(+) T cell responses also were significantly enhanced, indicating that PD-L1 has a role in limiting the expansion or survival of CD8(+) T cells. Studies using the myelin oligodendrocyte model of experimental autoimmune encephalomyelitis showed that PD-L1 on T cells and in host tissues limits responses of self-reactive CD4(+) T cells in vivo. PD-L1 deficiency converted the 129S4/SvJae strain from a resistant to experimental autoimmune encephalomyelitis-susceptible strain. Transfer of encephalitogenic T cells from wild-type mice into PD-L1(-/-) recipients led to exacerbated disease. Disease was even more severe in PD-L1(-/-) recipients of PD-L1(-/-) T cells. These results demonstrate that PD-L1 on T cells, APCs, and host tissue inhibits naïve and effector T cell responses and plays a critical role in T cell tolerance.

Abstract

We have previously shown that naive SJL (H-2(s)) mice, which are highly susceptible to myelin proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), have a very high frequency (1/20,000 CD4 T cells) of PLP(139-151)-reactive T cells in the naive repertoire. In this study, we examine the function of this endogenous PLP(139-151)-reactive repertoire in vivo and find that this repertoire encompasses the precursors of pathogenic T cells. Because SJL mice do not develop spontaneous EAE, we have explored the mechanisms that keep this autopathogenic repertoire in check and prevent the development of spontaneous autoimmunity. We crossed IL-4 and IL-10 deficiency onto the SJL background and analyzed the roles of these two immunoregulatory cytokines in regulating the size and effector function of the endogenous PLP(139-151)-reactive repertoire and development of autoimmune disease. We find that IL-10 is important in the homeostatic regulation of the endogenous PLP(139-151)-reactive repertoire in that it both limits the size of the repertoire and prevents development of effector autoaggressive T cells. SJL IL-10(-/-) mice with high numbers of PLP(139-151)-specific precursors in the repertoire did not develop spontaneous EAE, but when they were injected with pertussis toxin, they showed atypical clinical signs of EAE with small numbers of typical mononuclear cell infiltrates predominantly in the meninges. EAE could be inhibited by prior tolerization of the mice with soluble PLP(139-151) peptide. These findings indicate that IL-10 may contribute to the regulation of the endogenous autoimmune repertoire.

Abstract

The transcription factors signal transducer and activator of transcription (STAT)1 and T-bet control the differentiation of interferon (IFN)-gamma-producing T helper type (Th)1 cells. Here we compare the role of T-bet and STAT1 in the initiation and regulation of experimental autoimmune encephalomyelitis (EAE), a disease initiated by Th1 cells. T-bet-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) were resistant to the development of EAE. This protection was also observed when T-bet(-/-) mice were crossed to the MOG-specific 2D2 T cell receptor transgenic strain. In contrast, although T-bet is downstream of STAT1, STAT1(-/-) mice were highly susceptible to EAE and developed more severe and accelerated disease with atypical neuropathologic features. The function of T-bet was dominant as mice deficient in both T-bet and STAT1 were also protected from EAE. CD4(+) CD25(+) regulatory T cells from these two mice strains were fully competent and do not explain the difference in disease susceptibility. However, enhanced EAE in STAT1(-/-) mice was associated with continued generation of IFN-gamma-producing Th1 cells and up-regulation of selective chemokines responsible for the increased recruitment of macrophages and neutrophils in the central nervous system. Although the two transcription factors, STAT1 and T-bet, both induce IFN-gamma gene transcription, our results demonstrate marked differences in their function in regulating pathogenic Th1 cell responses.

Abstract

Nonobese diabetic (NOD) mice develop multi-organ autoimmune diseases, including type 1 diabetes. We hypothesized that backcrossing the MHC region from SJL (H-2(s)) mice, which have an endogenous PLP(139-151)-reactive repertoire, onto the background of autoimmune-prone NOD mice would result in a mouse strain that is highly susceptible to experimental autoimmune encephalomyelitis (EAE). Unexpectedly, although we detected an endogenous PLP(139-151) repertoire in the NOD.S mice, they did not develop spontaneous EAE and were relatively resistant to PLP(139-151)-induced EAE when compared to SJL mice. This resistance was associated with lower production of proinflammatory cytokines and a decreased expansion of PLP(139-151)-specific CD4(+) T cells after immunization and restimulation with PLP peptide in vitro. V(beta) chain usage among PLP(139-151)-reactive T cells differed between SJL and NOD.S mice. Furthermore, NOD.S mice were resistant to the development of insulitis and cyclophosphamide-induced diabetes, but not sialadenitis. Altogether, even though NOD mice develop spontaneous autoimmune diseases, they become relatively resistant to induction of EAE even when they express the EAE-permissive class II molecule I-A(s). Our data show that certain combinations of otherwise susceptibility-conferring MHC and non-MHC genes can mediate autoimmune-disease resistance when they are paired together. These findings do not support the "shared autoimmune gene" hypothesis.

Abstract

Linkage analysis and congenic mapping in NOD mice have identified a susceptibility locus for type 1 diabetes, Idd5.1 on mouse chromosome 1, which includes the Ctla4 and Icos genes. Besides type 1 diabetes, numerous autoimmune diseases have been mapped to a syntenic region on human chromosome 2q33. In this study we determined how the costimulatory molecules encoded by these genes contribute to the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). When we compared levels of expression of costimulatory molecules on T cells, we found higher ICOS and lower full-length CTLA-4 expression on activated NOD T cells compared with C57BL/6 (B6) and C57BL/10 (B10) T cells. Using NOD.B10 Idd5 congenic strains, we determined that a 2.1-Mb region controls the observed expression differences of ICOS. Although Idd5.1 congenic mice are resistant to diabetes, we found them more susceptible to myelin oligodendrocyte glycoprotein 35-55-induced EAE compared with NOD mice. Our data demonstrate that higher ICOS expression correlates with more IL-10 production by NOD-derived T cells, and this may be responsible for the less severe EAE in NOD mice compared with Idd5.1 congenic mice. Paradoxically, alleles at the Idd5.1 locus have opposite effects on two autoimmune diseases, diabetes and EAE. This may reflect differential roles for costimulatory pathways in inducing autoimmune responses depending upon the origin (tissue) of the target Ag.

Abstract

The immune events that take place in the central nervous system (CNS) during cryptococcal infection are incompletely understood. We used competitive reverse transcription-PCR to delineate the time course of the local expression of mRNAs encoding a variety of cytokines and inducible nitric oxide synthase (iNOS) during progressive murine cryptococcal meningoencephalitis and assessed the CNS inflammatory response using immunohistochemistry. Interleukin 18 (IL-18), transforming growth factor beta1, and IL-12p(40) mRNAs were constitutively expressed in the brains of infected and uninfected mice; IL-2 mRNA was not detected at any time. Increased levels of transcripts corresponding to IL-1 alpha, tumor necrosis factor alpha (TNF-alpha), and iNOS were detected as early as day 1 postinfection, with TNF-alpha rising by approximately 30-fold and iNOS increasing by approximately 5-fold by day 7. Each remained at these levels thereafter. IL-4, IL-6, and gamma interferon transcripts were detected on day 5, and IL-1 beta and IL-10 transcripts were detected beginning on day 7. Once detected, each remained at a relatively constant level through 28 days of infection. This cytokine profile does not suggest a polarized Th1 or Th2 response. Immunohistochemistry did not reveal inflammatory infiltrates before day 7, despite the presence of cryptococci. Intraparenchymal abscesses with inflammatory cells in their peripheries were found beginning on day 10. The infiltrates were comprised primarily of cells expressing CD4, CD8, or CD11b; low numbers of cells expressing CD45R/B220 were also present. The persistence of Cryptococcus observed in the CNS may result from an ineffective immune response, perhaps owing to an insufficient anticryptococcal effector function of endogenous glial cells resulting from competing pro- and anti-inflammatory cytokines. These data detail the immune response in the brain and could be important for the future design of specific immunomodulatory therapies for this important opportunistic infection.

Abstract

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells traffic into the CNS and induce disease. Infiltration of these pathogenic T cells into the CNS has been correlated with the expression of the chemokine IFN-inducible protein (IP)10/CXC chemokine ligand (CXCL)10, a chemoattractant for activated T cells, and its receptor CXCR3, in the CNS of both MS patients and mice with EAE. In the present study, we report that targeted deletion of IP-10 did not diminish the expression, severity, or histopathology of EAE induced by active immunization with 100 micro g of myelin oligodendrocyte glycoprotein peptide (MOG)p35-55. However, we found that IP-10-deficient mice had a lower threshold for expression of disease compared with wild-type littermates. EAE induced by immunization with 5 micro g of MOGp35-55 resulted in more severe disease characterized by a greater number of CNS lesions and infiltrating mononuclear cells in IP-10-deficient mice compared with wild-type controls. IP-10-deficient mice immunized with MOGp35-55 demonstrated increased levels of IFN-inducible T cell alpha-chemokine/CXCL11 mRNA in the CNS and decreased levels of monokine induced by IFN-gamma/CXCL9 mRNA in draining lymph nodes, suggesting differential compensation for loss of IP-10 in lymphoid vs parenchymal tissue compartments. EAE in IP-10-deficient mice induced by low-dose immunization was associated with enhanced Ag-specific Th1 responses in the draining lymph node, which corresponded with diminished lymph node TGF-beta1 expression. Our data demonstrated that IP-10 was not required for the trafficking of pathogenic T cells into the CNS in EAE but played an unexpected role in determining the threshold of disease susceptibility in the periphery.

Abstract

Schwannomatosis is a newly described form of neurofibromatosis of unclear pathogenesis.We studied the NF2 locus on chromosome 22 in 7 tumor specimens resected from a 36-year-old man with schwannomatosis of the right ulnar nerve.Unrelated truncating NF2 gene mutations were detected in 4 tumor specimens. None of the NF2 mutations were present in the blood specimen. Loss of heterozygosity at the NF2 locus was seen in all tumors, and in every case the same allele was lost. Loss of distal chromosome 22 markers was variable. Fluorescence in situ hybridization results were consistent with monosomy 22 in 4 tumors and mitotic recombination or nondisjunction in 1.Molecular analysis of tumor specimens distinguishes schwannomatosis from other forms of neurofibromatosis. Further work is needed to understand the natural history and molecular biology of this condition.

Abstract

Adoptive transfer experiments using C57BL/6 mice lacking B7-1 and B7-2 as recipients of wt (wt) encephalitogenic T cells demonstrate a key role for B7 costimulation during the effector phase of experimental autoimmune encephalomyelitis (EAE). Following transfer of encephalitogenic T cells, B7-1/B7-2-deficient (-/-) recipients develop a transient and mild disease as compared to wt recipients. To understand the mechanism by which B7-1/B7-2 may influence the effector phase of EAE, we analyzed T cells, pro-inflammatory cytokines and chemokines within the CNS of wt and B7-1/B7-2-/- recipients at different times after adoptive transfer of activated myelin specific T cells. There was a marked decline in T cells and inflammatory mediators in the CNS of B7-1/B7-2-/- recipients by day 30 post transfer. B7-1/B7-2-/- mice developed more TUNEL+ apoptotic cells in the parenchyma and greater ratios of TUNEL+ cells/parenchymal foci than wt mice resulting in virtual disappearance of parenchymal foci. Therefore, without B7-1 and B7-2 costimulation in the target organ, there is increased T cell apoptosis and attenuation of inflammation. These results indicate that B7-1 and B7-2 provide critical costimulatory signals for sustaining survival of pathogenic T cells within the central nervous system parenchyma during the effector phase of EAE and suggest novel treatment approaches in the effector phase of autoimmune diseases.

Abstract

Multiple sclerosis (MS) is considered to be an autoimmune disease of the central nervous system (CNS) that in many patients first presents clinically as optic neuritis. The relationship of optic neuritis to MS is not well understood. We have generated novel T cell receptor (TCR) transgenic mice specific for myelin oligodendrocyte glycoprotein (MOG). MOG-specific transgenic T cells are not deleted nor tolerized and are functionally competent. A large proportion (>30%) of MOG-specific TCR transgenic mice spontaneously develop isolated optic neuritis without any clinical nor histological evidence of experimental autoimmune encephalomyelitis (EAE). Optic neuritis without EAE could also be induced in these mice by sensitization with suboptimal doses of MOG. The predilection of these mice to develop optic neuritis is associated with higher expression of MOG in the optic nerve than in the spinal cord. These results demonstrate that clinical manifestations of CNS autoimmune disease will vary depending on the identity of the target autoantigen and that MOG-specific T cell responses are involved in the genesis of isolated optic neuritis.

Abstract

Nuclear factor-kappaB (NFkappaB) is a transcription factor that is activated after cerebral ischemia. NFkappaB activation leads to the expression of many inflammatory genes involved in the pathogenesis of stroke. The authors previously showed that mild hypothermia is protective even when cooling begins 2 h after stroke onset. In the present study, they examined the influence of hypothermia on NFkappaB activation. Rats underwent 2 h of transient middle cerebral artery occlusion. Brains were cooled to 33 degrees C immediately after or 2 h after occlusion, and maintained for 2 h. After normothermic ischemia (brain temperature at 38 degrees C), NFkappaB cytoplasmic expression, nuclear translocation, and binding activity were observed as early as 2 h in the ischemic hemisphere and persisted at 24 h. Hypothermia decreased NFkappaB translocation and binding activity but did not alter overall expression. Hypothermia also affected the levels of NFkappaB regulatory proteins by suppressing phosphorylation of NFkappaB's inhibitory protein (IkappaB-alpha) and IkappaB kinase (IKK-gamma) and decreasing IKK activity, but did not alter overall IKK levels. Hypothermia suppressed the expression of two NFkappaB target genes: inducible nitric oxide synthase and TNF-alpha. These data suggest that the protective effect of hypothermia on cerebral injury is, in part, related to NFkappaB inhibition due to decreased activity of IKK.

Abstract

Given the greater than 90% lethality of clinical central nervous system (CNS) aspergillosis despite current therapies, there is a need for an animal model to study therapeutic strategies. We previously established a model of CNS aspergillosis by intracerebral infection and report here the results of treatment with the two therapies with the greatest clinical experience, i.e., treatments with amphotericin B (AMB) and itraconazole (ITZ). Mice were given cyclophosphamide to produce pancytopenia. AMB was given intraperitoneally (i.p.; 3 mg/kg of body weight) or intravenously (i.v.; 0.8 mg/kg) once daily. ITZ in cyclodextrin was given by gavage once daily at a dose of 100 mg/kg or twice daily at 50 mg/kg. Treatments were started at day 1 postinfection and given for 10 days. At day 15, survivors were euthanatized. Ninety percent of the mice given no treatment died by day 6, and 100% died by day 10. Mice treated with AMB either i.p. or i.v. had 40% survival. Mice treated with ITZ either once or twice per day had a median survival time of 10 days, compared with 4 days for control animals, but a survival rate of only 10%. AMB and ITZ prolonged survival (P, <0.0001 to <0.05) compared with controls. Brains from surviving mice had CFU of Aspergillus fumigatus. This model can be used to compare newer antifungals and to study combination therapy or immunotherapy to find better therapeutic alternatives.

Abstract

We have previously reported studies of gene therapy using a neurotropic herpes simplex viral (HSV) vector system containing bipromoter vectors to transfer various protective genes to neurons. Using this system in experimental models of stroke, cardiac arrest, and excitotoxicity, we found that it is possible to enhance neuron survival against such cerebral insults by overexpressing genes that target various facets of injury. Among the genes we studied, the anti-apoptotic protein BCL-2 improved neuron survival following various insults, and was protective even when administered after stroke onset. BCL-2 is thought to protect cells from apoptotic death by preventing cytochrome c release from the mitochondria and subsequent caspase activation. We and others have established that cooling the brain by a few degrees markedly reduces ischemic injury and improves neurologic deficits in models of cerebral ischemia and trauma. This hypothermic neuroprotection is also associated with BCL-2 upregulation in some instances. Furthermore, hypothermia suppresses many aspects of apoptotic death including cytochrome c release, caspase activation, and DNA fragmentation. Here we show that two different kinds of protective therapies, BCL-2 overexpression and hypothermia, both inhibit aspects of apoptotic cell death cascades, and that a combination treatment can prolong the temporal therapeutic window for gene therapy.

Abstract

Matrix metalloproteinase (MMP)-9 is produced by the central nervous system and inflammatory cells in a variety of inflammatory conditions in both animals and humans. MMP-9 promotes inflammation, breakdown of the blood-brain barrier, and vasculitis. Because vasculitis is seen frequently in patients with coccidioidal meningitis (CM), this study evaluated the presence of MMP-9 within the cerebrospinal fluid (CSF) of rabbits infected intracisternally with Coccidioides immitis arthroconidia. Infected rabbits demonstrated systemic and neurological sequelae to infection, including CSF pleocytosis. Levels of MMP-9 within CSF were assayed by use of zymography and compared with MMP-2 levels, which served as an internal control. Elevated levels of MMP-9 were detectable by day 3, continued to increase through day 10, and declined by day 15 after infection. MMP-9 may contribute to inflammation and vasculitis in this animal model. Future work can focus on evaluation of MMP inhibitors, to gain a better perspective of the role of this MMP in CM.

Abstract

B7 costimulatory molecules play an important role in inducing autoimmunity, tumor immunity, and transplant rejection, and therapeutic manipulation of B7 is being investigated in human diseases. To determine whether B7 costimulation is essential for inducing autoimmunity on different genetic backgrounds, we backcrossed B7.1/B7.2 deficient ((-/-)) mice on to the C57BL/6 (B6) and SJL backgrounds and induced experimental autoimmune encephalomyelitis (EAE) in these mice. B7.1/B7.2(-/-) mice on the B6 background were resistant to EAE induced with MOG 35-55, whereas the SJL B7.1/B7.2(-/-) mice were susceptible to PLP 139-151 or PLP 178-191-induced EAE. The SJL B7.1/B7.2(-/-) mice had a qualitatively different lesion pattern in that they showed increased white matter vacuolation compared to wild-type SJL mice when immunized with either PLP 139-151 or PLP 178-191. (B6xSJL)F1 B7.1/B7.2(+/+) mice were susceptible to EAE whereas (B6xSJL)F1 B7.1/B7.2(-/-) mice were resistant to EAE induced with either encephalitogenic peptide. Thus, genetic background determines the B7 requirement for inducing autoimmunity. These data have important implications for developing B7-based immunotherapies for human diseases.

Abstract

Central nervous system (CNS) Aspergillus infection has a mortality rate in humans that approaches 95%. Because no animal models are available for studying this infection, we sought to develop a murine model of CNS aspergillosis. Inconsistent data were obtained for nonimmunosuppressed CD-1, C57BL/6, and DBA/2N mice after infection by midline intracranial injection of Aspergillus fumigatus. CD-1 mice given cyclophosphamide to produce immunosuppression had continuous pancytopenia. Dose-finding studies in CD-1 mice showed that infection with 5 x 106 conidia/mouse consistently caused 100% mortality by day 5-8; no mice died before day 3. Histologic examination of samples of brain tissue showed focal abscesses containing Aspergillus hyphae. Fungus burdens in brain were higher than those in other organs, although Aspergillus disseminated to the kidneys and the spleen. The model we established provides an opportunity to study immune responses to and therapeutic options for CNS disease in an immunologically defined, genetically manipulable, and inexpensive species.

Abstract

To elucidate mechanisms of endothelial cell (EC) dysfunction in CNS inflammatory responses and beneficial effects of interferon-beta (IFN-gamma) in multiple sclerosis (MS), we analyzed effects of individual and combinations of soluble inflammatory mediators on the intracellular localization of the EC tight junction-associated molecules zonula occludens-1 and -2 (ZO-1 and ZO-2) in human brain ECs. The cytoplasm in the majority of cells in control EC cultures was clear; ZO-1 and ZO-2 were localized peripherally near sites of cell contact and associated with submembranous cytoplasmic filaments. H2O2 induced reversible time- and concentration-dependent translocation of ZO-1 and ZO-2 to a random distribution within EC cytoplasm and retraction of EC borders. For low concentrations, these effects were accompanied by less prominent submembranous filaments but not by evidence of cytotoxicity, increased cell death or altered amounts of ZO-1. Tumor necrosis factor-beta induced similar alterations but interferon-y did not. Co-treatment with either cytokine increased H2O2 effects whereas IFN-beta reversed H2O2-induced effects. In control white matter samples, EC cytoplasm was clear and ZO-1 was located on cell borders. In inflammatory/demyelinating lesions, EC ZO-1 was diffuse, indicating that the alterations induced in vitro mimic those in active MS lesions. These findings suggest that in MS patients, IFN-beta treatment may counteract inflammatory mediator effects on CNS EC tight junction molecules, thereby preserving EC barrier function.

Abstract

The efficacy of intravenously administered liposomal amphotericin B (AmBisome [AmBi]) for the treatment of experimental coccidioidal meningitis was compared with those of oral fluconazole (FLC) and intravenously administered conventional amphotericin B (AMB). Male New Zealand White rabbits were infected by intracisternal inoculation of arthroconidia of Coccidioides immitis. Starting 5 days postinfection, animals received one of the following: 5% dextrose water diluent; AMB given at 1 mg/kg of body weight; AmBi given at 7.5, 15, or 22.5 mg/kg intravenously three times per week for 3 weeks; or oral FLC given at 80 mg/kg for 19 days. One week after the cessation of therapy, all survivors were euthanatized, the numbers of CFU remaining in the spinal cord and brain were determined, and histological analyses were performed. All AmBi-, FLC-, or AMB-treated animals survived and had prolonged lengths of survival compared with those for the controls (P < 0.0001). Treated groups had significantly lower numbers of white blood cells and significantly lower protein concentrations in the cerebrospinal fluid compared with those for the controls (P < 0.01 to 0.0005) and had fewer clinical signs of infection (e.g., weight loss, elevated temperature, and neurological abnormalities including motor abnormalities). The mean histological scores for AmBi-treated rabbits were lower than those for FLC-treated and control rabbits (P < 0.016 and 0.0005, respectively); the scores for AMB-treated animals were lower than those for the controls (P < 0.0005) but were similar to those for FLC-treated rabbits. All regimens reduced the numbers of CFU in the brain and spinal cord compared with those for the controls (P < or =0.0005). AmBi-treated animals had 3- to 11-fold lower numbers of CFU than FLC-treated rabbits and 6- to 35-fold lower numbers of CFU than AmB-treated rabbits. Three of eight animals given 15 mg of AmBi per kg had no detectable infection in either tissue, whereas other doses of AmBi or FLC cleared either the brain or the spinal cord of infection in fewer rabbits. In addition, clearance of the infection from both tissues was achieved in none of the rabbits, and neither tissue was cleared of infection in AMB-treated animals. Overall, these data indicate that intravenously administered AmBi is superior to oral FLC or intravenous AMB and that FLC is better than AMB against experimental coccidioidal meningitis. These data indicate that AmBi may offer an improvement in the treatment of coccidioidal meningitis. Additional studies are warranted.

Abstract

We and others previously reported that cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the severity of peptide-induced experimental autoimmune encephalomyelitis (EAE) in mouse strains that are inherently susceptible to the disease. In this report, we show that CTLA-4 engagement also controls disease susceptibility in BALB/c mice, a strain considered to be resistant to EAE induction. Although immunization of BALB/c mice with syngeneic spinal cord homogenate or an I-A(d)-binding myelin peptide antigen failed to result in EAE, immunization with either antigen preparation in conjunction with anti-CTLA-4 resulted in both clinical and histological EAE. CTLA-4 blockade also resulted in a preferential increase in the frequency of antigen-specific T cells secreting IFN-gamma. We conclude that CTLA-4 controls susceptibility in BALB/c mice by limiting the expansion of autoreactive T cells present in the periphery, suggesting a mechanism whereby CTLA-4 contributes to the maintenance of peripheral T cell tolerance to self antigens.

Abstract

Activation of naive CD4(+) T-helper cells results in the development of at least two distinct effector populations, Th1 and Th2 cells. Th1 cells produce cytokines (interferon (IFN)-gamma, interleukin (IL)-2, tumour-necrosis factor (TNF)-alpha and lymphotoxin) that are commonly associated with cell-mediated immune responses against intracellular pathogens, delayed-type hypersensitivity reactions, and induction of organ-specific autoimmune diseases. Th2 cells produce cytokines (IL-4, IL-10 and IL-13) that are crucial for control of extracellular helminthic infections and promote atopic and allergic diseases. Although much is known about the functions of these two subsets of T-helper cells, there are few known surface molecules that distinguish between them. We report here the identification and characterization of a transmembrane protein, Tim-3, which contains an immunoglobulin and a mucin-like domain and is expressed on differentiated Th1 cells. In vivo administration of antibody to Tim-3 enhances the clinical and pathological severity of experimental autoimmune encephalomyelitis (EAE), a Th1-dependent autoimmune disease, and increases the number and activation level of macrophages. Tim-3 may have an important role in the induction of autoimmune diseases by regulating macrophage activation and/or function.

Abstract

Extracellular matrix (ECM) alterations in the central nervous system (CNS) of multiple sclerosis (MS) patients result from blood-brain barrier breakdown, release and activation of proteases, and synthesis of ECM components. To elucidate their potential pathophysiologic roles, we analyzed expression of major CNS ECM proteoglycans (PGs) in MS and control CNS tissues. In active MS plaque edges, 3 CNS lecticans (versican, aggrecan, and neurocan) and dermatan sulfate PG were increased in association with astrocytosis; in active plaque centers they were decreased in the ECM and accumulated in foamy macrophages, suggesting that these ECM PGs are injured and phagocytosed along with myelin. In inactive lesions they were diminished and in normal-appearing white matter they showed heretofore-unappreciated abnormal heterogeneous aggregation. Phosphacan, an ECM PG abundant in both gray and white matter, was less markedly altered. Since in development the spaciotemporal expression of ECM PGs influences neurite outgrowth, cell migration, axon guidance, and myelination, these data suggest that 1) enhanced white matter lectican and dermatan sulfate PG expression in the pro-inflammatory milieu of expanding lesion edges contributes to their sharp boundaries and the failure of neuronal ingrowth; 2) decreases in plaque centers may preclude regeneration and repair; and 3) diffuse ECM PG damage relates to axon degeneration outside of overt lesions. Thus, ECM PG alterations are specific, temporally dynamic, and widespread in MS patients and may play critical roles in lesion pathogenesis and CNS dysfunction.

Abstract

Multiple sclerosis is a demyelinating disease, characterized by inflammation in the brain and spinal cord, possibly due to autoimmunity. Large-scale sequencing of cDNA libraries, derived from plaques dissected from brains of patients with multiple sclerosis (MS), indicated an abundance of transcripts for osteopontin (OPN). Microarray analysis of spinal cords from rats paralyzed by experimental autoimmune encephalomyelitis (EAE), a model of MS, also revealed increased OPN transcripts. Osteopontin-deficient mice were resistant to progressive EAE and had frequent remissions, and myelin-reactive T cells in OPN-/- mice produced more interleukin 10 and less interferon-gamma than in OPN+/+ mice. Osteopontin thus appears to regulate T helper cell-1 (TH1)-mediated demyelinating disease, and it may offer a potential target in blocking development of progressive MS.

Abstract

Mild hypothermia protects the brain against experimental ischemia, but the reasons are not well known. We examined whether the protective effects of mild hypothermia could be correlated with alterations in expression of Bcl-2, an anti-apoptotic protein in a rat model of transient global ischemia. Following 10 min of forebrain ischemia, hippocampal neurons were examined 72 h later for survival, expression of Bcl-2 family proteins and apoptosis. Intraischemic mild hypothermia was applied for 3 h (33 degrees C, isch-33) or normal body temperature was maintained (37 degrees C, isch-37). Survival of CA1 neurons was significantly improved in the isch-33 group compared to the isch-37 group (90 vs. 53% survival; P<0.01). The proportion of Bcl-2-positive cells among surviving CA1 neurons in the isch-33 group was increased compared to that of sham and isch-37 groups (P<0.01). Bax expression in CA1 was no different between sham and isch-33 groups, but was significantly decreased in isch-37 (P<0.05). TUNEL staining was positive in many isch-37 CA1 neurons, but absent in isch-33. Utilizing electron microscopy, more cells meeting criteria for apoptosis were observed in the isch-37 than isch-33. These data suggest that mild hypothermia attenuates apoptotic death, and that this protection may be related to increases in Bcl-2.

Abstract

Diffusion-weighted MRI (DWI) can detect early ischemic changes and is sometimes used as a surrogate neurological end point in clinical trials. Recent experimental stroke studies have shown that with brief periods of ischemia, some DWI lesions transiently reverse, only to recur later. This study examined the histological condition of the tissue during the period of DWI reversal.Rats underwent 30 minutes of middle cerebral artery occlusion followed by reperfusion. DWI images were obtained during ischemia and 3 to 5 hours, 1 day, and 7 days later. MRI scans were compared with histology (5 hours, n=5; 7 days, n=5) with the use of neuronal (microtubule-associated protein 2 [MAP2]) and astrocytic (glial fibrillary acidic protein [GFAP]) markers and heat-shock protein 72 (HSP72).DWI abnormalities reversed 3 to 5 hours after ischemia onset but recurred at 1 day. Four animals showed complete reversal of the initial DWI hyperintensity, and 6 showed partial reversal. When the 5-hour DWI was completely normal, there was significant loss of MAP2 immunoreactivity, comprising approximately 30% of the initial DWI lesion. However, GFAP staining revealed morphologically normal astrocytes. HSP72 immunoreactivity at 5 hours was extensive and corresponded to the initial DWI lesion.After brief ischemic periods, normalization of the DWI does not necessarily imply that the tissue is normal. Neurons already exhibit evidence of structural damage and stress. Normal GFAP staining suggests that other nonneuronal cell populations may partially compensate for altered fluid balances at the time of DWI reversal despite the presence of neuronal injury. These observations suggest that caution is warranted when relying solely on DWI for assessment of ischemic damage.

Abstract

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

Abstract

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is an inducible inflammatory and demyelinating disease of the central nervous system (CNS). Susceptibility to this disease is heritable and is demonstrated by the development of an ascending paralysis accompanied by a loss in body wt 2-3 weeks following immunization with proteins derived from CNS myelin. In a previous genetic analysis of susceptibility to EAE in a cross between susceptible SJL/J mice and resistant B10.S mice, we found suggestive evidence of linkage with disease susceptibility at the telomeric end of chromosome 2 and in the central region of chromosome 3. To define these associations more precisely and to investigate the genetic factors controlling measurable phenotypes of EAE, we performed a new analysis with a larger number of mice. The results now indicate that the chromosome 2 locus significantly influences EAE-related weight loss (P = 6.7 x 10(-5)) and that the chromosome 3 locus is linked with the development of paralysis. In addition, an intriguing inheritance pattern was revealed in which female backcross mice generated from B10.S female x (B10.S x SJL/J)F(1) male parents experienced significantly more EAE-related weight loss (P = 1.2 x 10(-4)) than females generated from F1 female x B10.S male parents. After controlling for this inheritance, a new locus at the centromeric end of chromosome 8 was identified that significantly influences both the development of paralysis (P = 8.2 x 10(-6)) and the incidence of CNS inflammation (P = 7.0 x 10(-5)) in EAE.

Abstract

The lipid formulation of amphotericin B, Amphotec (ABCD), has not been used intrathecally. After a single intrathecal dose or after four doses, conventionally formulated deoxycholate amphotericin B (AMB) (Fungizone) resulted in higher levels of amphotericin B in the cerebrospinal fluid of rabbits than did ABCD. Clinically and histologically, ABCD was about threefold less toxic than AMB after a single dose and 3- to 30-fold less toxic after multiple dosing. These data are encouraging for the potential use of ABCD as an intrathecal treatment.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the size, reactivity, and function of a primed pool of CD4(+) T cellsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKuhns, M. S., Epshteyn, V., Sobel, R. A., Allison, L. P.2000; 97 (23): 12711-12716

Abstract

We examined how cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates heterogeneous CD4(+) T cell responses by using experimental autoimmune encephalomyelitis (EAE), a CD4(+) T cell-mediated disease that is subject to regulation by CTLA-4. Disease incidence and severity were used as measures of in vivo CD4(+) T cell responses. The frequency, cytokine production, and reactivity of primed T cells were determined from animals immunized with proteolipid protein (PLP)-139-151 (disease agonist), PLP-Q (disease antagonist), or both peptides, and treated with control or anti-CTLA-4 antibody to analyze the responding population. CTLA-4 blockade exacerbated disease in PLP-139-151-primed animals and overcame disease antagonism in coimmunized animals, but did not permit disease induction in PLP-Q-primed animals. Experimental autoimmune encephalomyelitis enhancement was associated with increased frequencies of cytokine-producing cells and increased ratios of IFN-gamma to IL-4 secretors responsive to PLP-139-151. Priming with PLP-Q elicited IL-4 and IL-2, but not IFN-gamma secretors cross-reactive with PLP-139-151. Strikingly, CTLA-4 blockade was found to decrease rather than increase the frequencies of cross-reactive IL-4 and IL-2 secretors. Thus, CTLA-4 engagement limits the size, but increases the breadth, of reactivity of a primed pool of CD4(+) T cells, consequently regulating its function.

Abstract

A rabbit model of coccidioidal meningitis was used to compare the therapeutic efficacies of terbinafine (TBF) and fluconazole (FCZ). Hydrocortisone acetate-treated New Zealand White male rabbits were infected intracisternally with either 2.2 x 10(4) or 6.4 x 10(4) Coccidioides immitis arthroconidia. Oral treatment with polyethylene glycol 200 (PEG) twice daily (n = 8), TBF twice daily (n = 9; 200 mg/kg of body weight/day), or FCZ once daily (n = 8; 80 mg/kg/day) began on day 5 and continued for 21 days. Mean survival times were 20, 24, and 32 days for rabbits treated with PEG, TBF, and FCZ, respectively. All of the FCZ-treated animals (100%; P = 0.003), 56% of the TBF-treated animals (P = 0.4), and 25% of the PEG-treated animals survived the length of the study. Both FCZ and TBF were effective at reducing the incidence of paresis. Only FCZ was effective at reducing most neurological and systemic signs. FCZ treatments resulted in lower cerebrospinal fluid (CSF) protein concentrations and leukocyte counts and faster clearing of CSF fungal cultures compared with those for PEG-treated controls, but TBF treatments had no significant effect on these parameters. Neither drug affected CSF glucose levels. Mean serum TBF levels by bioassay were within the range of 3.5 to 6.2 microgram/ml at 1, 2, and 4 h postdosing and 0.35 to 7.0 microgram/ml at 14 h postdosing. No TBF was detected in CSF. Mean FCZ levels (24 to 25.5 h postdosing) by bioassay were 16.4 to 19.2 and 13.5 to 19.2 microgram/ml in serum and CSF, respectively. The reduction in the numbers of CFU in the spinal cord and brain was over 100-fold (P = 0.0005) in FCZ-treated animals and 2-fold (P = 0.2) in TBF-treated animals compared with those in PEG-treated animals. Histopathologic severity (semiquantitative scoring system) was significantly attenuated by FCZ treatment (P = 0. 05) and was slightly attenuated by TBF treatment compared with that for the controls. In conclusion, TBF appeared to have a slight effect on survival, histology, and reduction of the numbers of CFU in tissue; however, these effects were not significant. FCZ was effective at controlling coccidioidal meningitis.

Abstract

IL-10 is associated with a Th2 response, down-regulation of a Th1 response and macrophage activation. We assessed the role of IL-10 during systemic infection with Aspergillus fumigatus. Systemic aspergillosis was established in female C56B1/6 IL-10(-/-) (KO) and wild-type (WT) C57B1/6 mice by i.v. administration of 1 x 10(5)-6 x 10(5) conidia of A. fumigatus. In two experiments, KO survived longer than did WT (P < 0.001). Determination of fungal burdens in the kidneys and brain showed that KO carried significantly lower burdens in both organs than did WT on day 3 (P < 0.001). Semiquantitative histological analyses showed fewer inflammatory foci/mm2 in brain and kidneys of KO than WT (P < 0.03 and < 0.001, respectively) and that extent of infection and associated tissue injury were greater in WT. Although beneficial in some bacterial infections, exogenous IL-10 has been shown deleterious in models of fungal infection. Our data indicate IL-10 is deleterious during systemic aspergillosis infection, increasing the host susceptibility to lethal infection. We speculate this might be related to greater Th2 or lesser Th1 responses, or down-regulation of macrophage responses, in WT compared with KO.

Abstract

Different encephalitogenic peptides can induce two distinct experimental autoimmune encephalomyelitis (EAE) phenotypes in different mouse strains. To determine whether different peptides induce distinct phenotypes in genetically identical mice, parental strain and (SJLXC3H/HeJ)F1 mice were sensitized with myelin proteolipid protein peptide p139-151 or p215-232. p139-151 was non-encephalitogenic in C3H/HeJ mice and p215-232 was non-encephalitogenic in SJL mice. p139-151 induced typical acute EAE in SJL and F1 mice with most CNS inflammatory/demyelinating lesions located in the spinal cord. p215-232 induced mild clinical disease in only two of 10 C3H/HeJ mice; in 11 of 13 F1 mice (85%) it induced a disease spectrum that included typical paralytic acute EAE with a predominance of spinal cord lesions and later-onset mild EAE with predominance of brain stem/cerebellar lesions. Thus, the EAE phenotype induced in F1 mice by one encephalitogen, e.g. p139-151, can be the same as that induced in the susceptible parent. However, other encephalitogenic peptides, e.g. p215-232, may induce a broad range of heterogeneous EAE phenotypes in syngeneic mice. These data indicate that in some encephalitogenic responses, epigenetic factors influence EAE incidence, time of onset, severity, neurological signs and CNS lesion distribution.

Abstract

Histoplasma capsulatum is an important fungal pathogen in immunocompromised hosts, including AIDS patients. Experimental evidence suggests interferon-gamma (IFN) plays a role in host defense against H. capsulatum. In these studies we sought to demonstrate the importance of IFN in innate resistance to systemic histoplasmosis. The possible exacerbation of infection in BALB/c mice was assessed by administering 200 microg of hamster anti-IFN antibody prior to infection with H. capsulatum (2 x 10(6) yeasts, i.v.) and by comparing the severity of infection between BALB/c IFN gene knockout mice (GKO) and congenic control animals. In two separate studies, we found that anti-IFN treatment caused a dramatic loss of resistance to lethal infection and resulted in earlier mortality of IFN-depleted animals compared with normal IgG or no treatment (P<0.001). GKO mice were significantly (P<0.001) more susceptible to lethal infection than were control animals, and histological studies corroborated this. These studies clearly demonstrate that IFN is a vital part of the host's innate resistance to systemic infection with H. capsulatum and provide an additional rationale for studying IFN as an immunomodulatory therapeutic for the treatment of this disease.

Abstract

Coccidioidal meningitis is a devastating disease that requires long-term therapy with little hope of cure. A rabbit model of coccidioidal meningitis was used to compare the therapeutic efficacies of fluconazole (FCZ) and itraconazole (ITZ). Hydrocortisone-treated male New Zealand white rabbits were infected intracisternally with 5.0x10(4) to 5.4x10(4) arthroconidia of Coccidioides immitis. Oral treatment with polyethylene glycol 200 (PEG) (n = 9), FCZ (n = 8; 80 mg/kg of body weight/day), or ITZ (n = 8; 80 mg/kg/day) began 5 days after infection and continued for 28 consecutive days. Both FCZ and ITZ reduced the number of CFU of C. immitis organisms in the spinal cord and brain compared with the number in PEG-treated animals (P< or =0.003), but the results for FCZ and ITZ were not different from each other. Histopathologic severity (semiquantitative scoring system by an observer blinded to treatment) was equally reduced in both FCZ and ITZ treatment groups compared with that in controls (P< or =0.0004). Both treatments resulted in lower cerebrospinal fluid (CSF) protein concentrations and leukocyte counts and faster clearing of C. immitis from CSF compared with the results for PEG-treated controls. Neither drug affected CSF glucose levels. Both compounds were effective at reducing neurological and systemic signs and extending survival (P< or =0.014). FCZ was more effective at reducing head and body shakes, posture changes, and incontinence; ITZ was more effective at reducing continuous fever. Mean levels of FCZ and ITZ in the serum and CSF were determined by bioassay; at 17 to 26 h postdosing, levels were 28.1 to 40.0 and 22.4 to 29.9 microg/ml, respectively, for FCZ and 0.77 to 2.51 and 0 microg/ml, respectively, for ITZ. The sera of most animals developed antibody to C. immitis, but azole treatment attenuated antibody development in CSF and its titer. In conclusion, both FCZ and ITZ were efficacious, but neither was curative in a rabbit model of coccidioidal meningitis.

Fulminant spontaneous autoimmunity of the central nervous system in mice transgenic for the myelin proteolipid protein-specific T cell receptorPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAWaldner, H., Whitters, M. J., Sobel, R. A., Collins, M., Kuchroo, V. K.2000; 97 (7): 3412-3417

Abstract

Proteolipid protein (PLP)-139-151 is the dominant encephalitogenic peptide that induces experimental autoimmune encephalomyelitis (EAE) in SJL (H-2(s)) mice. To examine the contribution of T cell receptor (TCR) specificity in the induction of EAE, we generated transgenic mice expressing the rearranged TCR genes from an encephalitogenic or a nonencephalitogenic PLP-139-151/I-A(s)-specific T cell clone. Both types of transgenic lines developed spontaneous EAE, but, remarkably, the lines expressing the TCR from the nonencephalitogenic clone showed increasingly higher frequencies of disease (60-83%) in progressive SJL backcrosses and could not be propagated on the susceptible background. The T cells from the transgenic mice were not tolerized, because they responded vigorously to the antigen in vitro and mediated EAE when the mice were immunized with antigen. Besides being the only description of a TCR transgenic mice for the PLP-139-151/I-A(s) epitope, the results demonstrate that the TCR from a nonencephalitogenic PLP-specific T cell clone can induce autoimmune disease when expressed appropriately in vivo.

Abstract

To determine the contribution of B cells to brain myelin injury in Semliki Forest Virus (SFV) encephalomyelitis, normal C57BL/6 (B6) and B-cell-deficient (C57BL/6-tm1Cgn) B6 mice were infected with SFV. The peak of clinical disease, i.e., the time at which the greatest proportions of mice had moderate to severe clinical signs, appeared earlier in B6 mice [day 7 postinfection (pi)] than in B-cell-deficient mice (day 21 pi). By flow cytometry, no clear differences were found in the percentages of CD3(+)CD4(+) T cells in the brains of B6 and B-cell-deficient mice. However, by day 21 pi, percentages of CD3(+)CD8(+) T cells were greater in brains of B-cell-deficient than in those of B6 mice. On day 21 pi, percentages of CD19(+) B cells were maximal in B6 mice, but B cells were absent in B-cell-deficient mice at all time points. Sera obtained from B6 mice showed antibody responses to SFV, to SFV E2 peptides p137-151 and p115-133, and to peptides of myelin oligodendrocyte glycoprotein p18-32 and myelin basic protein (MBP) p64-75. Sera obtained from B-cell-deficient mice showed minimal or no reactivity to SFV, E2, or myelin peptides. CNS inflammatory and PAS-positive macrophage foci were maximal on days 7-14 pi in all mice. Additionally, B6 mice had brain white matter vacuolation, whereas B-cell-deficient mice did not. These data suggest that brain infiltrating B cells and anti-myelin antibodies contribute to myelin injury in SFV encephalomyelitis.

Abstract

LY303366 is a semisynthetic derivative of the echinocandin class. During preclinical studies, lethal toxicity was observed in DBA/2 mice pretreated with a cortisone acetate dose followed by treatment with LY303366 at doses ranging from 12.5 to 50 mg/kg of body weight/day given intraperitoneally (i.p.). In the cortisone-treated, uninfected controls, 90% given LY303366 at 50 mg/kg died. Deaths occurred only in steroid-treated mice. In additional experiments, uninfected DBA/2 and CD-1 mice were pretreated with different glucocorticoids. Dosages were adjusted for comparative potency with cortisone and were given at one, two, or five times the equivalent cortisone dosage of 5 mg prior to treatment with LY303366 at 25 mg/kg/day given i.p. Lethal toxicity occurred in DBA/2 mice given hydrocortisone (1x or 2x), triamcinolone (1x or 5x), and cortisone. However, no mice pretreated with 1x or 5x dexamethasone died. In CD-1 mice, deaths occurred only in those given 5x triamcinolone; three of five died 2 days after the cessation of 10 days of LY303366 treatment. The causes of the deaths and why inbred DBA/2 mice are more sensitive than outbred CD-1 mice to the combined lethal effects of LY303366 and some glucocorticoids could not be determined histologically and remain unexplained. This is the first report of this toxicity of combination glucocorticoids and LY303366. Whether a similar toxicity might apply to the other compounds in the echinocandin class of antifungals and the species specificity require additional study. In addition, the clinical relevance of these observations in steroid-treated patients to the clinical safety of LY303366 and other echinocandins needs to be determined.

Abstract

The importance of B7 costimulation in regulating T cell expansion and peripheral tolerance suggests that it may also play a significant regulatory role in the development of autoimmune disease. It is unclear whether B7 costimulation is involved only in the expansion of autoreactive T cells in the periphery, or if it is also required for effector activation of autoreactive T cells in the target organ for mediating tissue injury and propagating autoimmune disease. In this study, the role of B7-CD28 costimulation and the relative importance of B7 costimulators for the induction and effector phases of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide were examined. Wild-type, B7-1/B7-2-deficient mice, or CD28-deficient C57BL/6 mice were immunized with MOG 35-55 peptide. Mice lacking both B7-1 and B7-2 or CD28 showed no or minimal clinical signs of EAE and markedly reduced inflammatory infiltrates in the brain and spinal cord. However, mice lacking either B7-1 or B7-2 alone developed clinical and pathologic EAE that was comparable to EAE in wild-type mice, indicating overlapping functions for B7-1 and B7-2. Resistance to EAE was not due to a lack of induction of T helper type 1 (Th1) cytokines, since T cells from B7-1/B7-2(-/-) mice show reduced proliferative responses, but greater interferon gamma production compared with T cells from wild-type mice. To study the role of B7 molecules in the effector phase of the disease, MOG 35-55-specific T lines were adoptively transferred into the B7-1/B7-2(-/-) and wild-type mice. Clinical and histologic EAE were markedly reduced in B7-1/B7-2(-/-) compared with wild-type recipient mice. These results demonstrate that B7 costimulation has critical roles not only in the initial activation and expansion of MOG-reactive T cells, but also in the effector phase of encephalitogenic T cell activation within the central nervous system.

A selective role of calcineurin A alpha in synaptic depotentiation in hippocampusPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAZhuo, M., Zhang, W., Son, H., Mansuy, I., Sobel, R. A., Seidman, J., Kandel, E. R.1999; 96 (8): 4650-4655

Abstract

Pharmacological studies have suggested that long-term potentiation (LTP) and long-term depression (LTD) and depotentiation, three forms of synaptic plasticity in the hippocampus, require the activity of the phosphatase calcineurin. At least two different isoforms of calcineurin are found in the central nervous system. To investigate whether all of these forms of synaptic plasticity require the same isoforms of calcineurin, we have examined LTD, depotentiation, and LTP in mice lacking the predominant calcineurin isoform in the central nervous system, Aalpha-/- mice. Depotentiation was abolished completely whereas neither LTD nor LTP were affected. These studies provide genetic evidence that the Aalpha isoform of calcineurin is important for the reversal of LTP in the hippocampus and indicate that depotentiation and LTD operate through somewhat different molecular mechanisms.

Abstract

To determine if central nervous system (CNS) microvessel endothelial cells express class II major histocompatibility complex (MHC) molecules in early demyelinating lesions in humans, cerebral white matter (WM) biopsies from patients with acute inflammatory/demyelinating conditions, including 4 with multiple sclerosis (MS), were immunostained for class II MHC and other antigens. Eight of 9 biopsies showed focal MHC class II-positive endothelial cells; there were none in the CNS of 1 of the MS patients at autopsy. There were more vessels with class II-positive endothelial cells in areas with intact WM and gliosis than in areas with active demyelination or control WM; class II-positive endothelial cells in small venules and capillaries were adjacent to transmigrating and perivascular CD4-positive cells. By immunoelectron microscopy, class II molecules were localized to vesicles in endothelial cell cytoplasm, suggesting the potential for antigen processing. Perivascular cells, parenchymal microglia, mononuclear cells and the perinuclear cytoplasm but not the processes of astrocytes were also class II-positive. These data indicate that in acute CNS inflammatory/demyelinating lesions, endothelial cells focally and apparently transiently express class II MHC molecules. This expression implies potential antigen-specific interactions, immunoregulatory or signalling functions in endothelial cells, or it may render them susceptible to CD4-positive cell-mediated cytotoxicity. Thus, class II-positive endothelial cells may have pivotal immunologic roles in initial stages of T cell responses in human CNS WM, particularly in acute MS lesions.

Abstract

Semliki Forest Virus (SFV) induces an encephalomyelitis followed by demyelination in the brains of C57Bl6/J (B6) mice. To investigate the role of molecular mimicry in the pathogenesis of postviral demyelination, alignment algorithms were used and amino acid homologies between immunogenic epitopes of SFV and myelin autoantigens, myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) were identified. Immunization of B6 mice with SFV proteins induced significant lymphocyte proliferation to SFV E2 peptides and to MOG peptide, 18-32 (which had molecular mimicry with E2 115-129), but not to MBP or PLP peptides. Both MOG 18-32 and E2 115-129, induced a later-onset chronic EAE-like disease that correlated with the presence of multifocal vacuolation in the CNS white matter. This histopathology was reminiscent of the secondary demyelination seen following SFV infection. Serum antibody responses to the peptides appeared late after immunizations and some samples cross-reacted with other myelin peptides, as well as with the mimicked MOG peptides. These findings suggest that following a CNS viral infection, antibody response to an epitope of virus that exhibits molecular mimicry with a peptide of MOG may contribute to autoimmune mediated injury to CNS myelin.

Abstract

The importance of natural killer (NK) cells in the resistance to herpes simplex virus type 1 (HSV-1), a common infection of immunocompromised patients, is unclear. Previous data on the role of NK cells in murine HSV-1 infection has been contradictory. Adoptive transfer studies suggested that NK cells mediated resistance to HSV-1, but in vivo depletion approaches demonstrated that NK cells were not important. We studied the course of HSV-1 infection after intranasal (i.n.) inoculation of E26 mice (lacking NK and T cells), T cell knockout (T cell ko) mice (lacking T cells only), or normal control mice. The E26 mice showed greater mortality and an impaired ability to clear virus from lung and brain compared to T cell ko mice and control mice, and had severe necrotizing HSV-1 encephalitis. Therefore, the data support the hypothesis that NK cells play an important role in the natural defense of murine HSV-1 infection.

Abstract

Coccidiodal meningitis is a devastating complication of disseminated coccidioidomycosis. An animal model of this infection could enhance understanding of the pathogenesis of the disease and lead to improvements in therapy. A rabbit model of central nervous system infection simulating human disease was established using a blind cisternal tap technique to inoculate 4 x 10(3)-1 x 10(6) arthroconidia of Coccidioides immitis into the cisterna magna. Systemic, neurologic, and histopathologic findings of meningitis were observed in all rabbits, but an inoculum of 2 x 10(4) arthroconidia produced a chronic illness in which meningeal endarteritis obliterans was consistently observed. Serial sampling of cerebrospinal fluid demonstrated an inflammatory response. Growth of C. immitis was demonstrated by quantitative fungal culture from brains and proximal spinal cords.

Abstract

Experimental autoimmune encephalomyelitis (EAE) and other organ-specific autoimmune diseases are induced by autoantigen-specific Th1 cells. In contrast, transfer of autoantigen-reactive Th2 cells that produce IL-4 and IL-10 can prevent and/or reverse EAE. The relative roles of these two Th2 cytokines in the regulation of EAE has not been evaluated. Utilizing IL-4 and IL-10 knockout mice deficient for these cytokines and IL-10 and IL-4 transgenic mice overexpressing these cytokines, we demonstrate that IL-10-deficient mice (IL-10(-/-)) are more susceptible and develop a more severe EAE when compared with IL-4-deficient mice (IL-4(-/-)) or wild-type mice. T cells from IL-10(-/-) mice exhibit a stronger Ag-specific proliferation, produce more proinflammatory cytokines (IFN-gamma and TNF-alpha) when stimulated with an encephalitogenic peptide, and induce very severe EAE upon transfer into wild-type mice. In contrast, while IL-4 transgenic mice develop similar disease compared with their nontransgenic littermates, mice transgenic for IL-10 are completely resistant to the development of EAE. Taken together, our data suggest that IL-10 plays a more critical role in the regulation of EAE by regulating autopathogenic Th1 responses.

Abstract

Cross-reactivity with environmental antigens has been postulated as a mechanism responsible for the induction of autoimmune disease. Experimental autoimmune encephalomyelitis is a T cell-mediated autoimmune disease model inducible in susceptible strains of laboratory animals by immunization with protein constituents of myelin. We used myelin proteolipid protein (PLP) peptide 139-151 and its analogues to define motifs to search a protein database for structural homologues of PLP139-151 and identified five peptides derived from microbial Ags that elicit immune responses that cross-react with this self peptide. Exposure of naive SJL mice to the cross-reactive environmental peptides alone was insufficient to induce autoimmune disease even when animals were treated with Ag-nonspecific stimuli (superantigen or LPS). However, immunization of SJL mice with suboptimal doses of PLP139-151 after priming with cross-reactive environmental peptides consistently induced experimental autoimmune encephalomyelitis. Furthermore, T cell lines from mice immunized with cross-reactive environmental peptides and restimulated in vitro with PLP139-151 could induce disease upon transfer into naive recipients. These data suggest that expansion by self Ag is required to break the threshold to autoimmune disease in animals primed with cross-reactive peptides.

Abstract

Laminin, a major glycoprotein component of vessel basement membranes, is recognized by beta1- and beta3-integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions, integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions, endothelial cell VLA-6 and beta1 subunits were decreased compared to controls whereas alpha(v) subunit and VLA-1 were increased. In chronic inactive lesions beta1, VLA-6 and alpha(v) were the same as controls but VLA-1 remained increased. Alpha3 subunit was constant in all samples. By immunoelectron microscopy VLA-1, VLA-6, beta1, and laminin were distributed throughout endothelial cells; alpha(v) was adjacent to and on luminal surfaces; alpha(v) and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell beta1/VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and blood-brain barrier breakdown. Alpha(v) and VLA-1 on intercellular junctions may participate in re-establishing vessel integrity after leukocyte migration. Luminal surface alpha(v) also likely binds intraluminal ligands and cells. In chronic inactive plaques persistently elevated endothelial cell VLA-1 correlates with long-standing endothelial cell and blood-brain barrier dysfunction.

Abstract

Peripheral macrophages infiltrating the central nervous system and resident microglia phagocytize myelin in cell-mediated demyelinating diseases, including experimental autoimmune encephalomyelitis and multiple sclerosis. A cascade of cytokines is believed to modulate the immunological sequence of events occurring in these conditions, and several of these mediate their effects through the protein kinase C pathway. Therefore, we compared the effects of phorbol myristate acetate (PMA), an activator of protein kinase C, on various functions of cultured macrophages and microglia. PMA at moderate concentrations induced apoptosis in macrophages, and this process appeared to be increased in the presence of myelin. In contrast, microglia were activated by PMA, and greatly increased their phagocytosis of myelin. Control macrophages released a considerable amount of proteolytic activity into the medium, as measured by the breakdown of myelin basic protein, and in the process of undergoing apoptosis from PMA-treatment, even higher amounts were released. The enzyme activity in control macrophage medium was inhibited mainly by PMSF and calpain inhibitors, while that from PMA-treated macrophages was inhibited by calpain inhibitors only. An ICE inhibitor was ineffective in inhibiting activity in medium from PMA-treated cells undergoing apoptosis. Medium from microglia contained very little proteolytic activity, and this was not increased by PMA. Cultured macrophages showed little evidence of oxygen free radical release as measured by the TBARS procedure, and PMA had no effect. Microglia, on the other hand, produced higher levels of reactive oxygen species, with a further increase of 18% by PMA. Thus major functions of these phagocytic cells appear to be modulated by the protein kinase C pathway, although the two cell types show very different responses to an activator of this signal.

Abstract

An altered peptide ligand (analog) of the encephalitogenic epitope of proteolipid protein residues 139-151 (p139-151) in which residues 144 and 147 are substituted with leucine and arginine, respectively (LR), protects from clinical but not histological experimental allergic encephalomyelitis (EAE). To understand in situ events associated with this protection, T cells from brains of mice immunized with either native p139-151, the analog LR or a combination of the two were isolated and characterized. High proportions of cells from co-immunized mice (38%) and LR-immunized mice (58%) reacted to both p139-151 and LR, whereas fewer cells from p139-151 immunized mice (7%) were cross-reactive. T cell clones derived from brains of LR- and co-immunized mice were also cross-reactive in vitro. By reverse transcriptase-based polymerase chain reaction, higher levels of TGF-beta mRNA, and lower levels of TNF-alpha and IFN-gamma mRNA were found in the central nervous system (CNS) tissue of LR and co-immunized mice. Immunohistochemistry demonstrated greater TGF-beta immunoreactivity in CNS inflammatory foci in co-immunized and LR-immunized mice. There were no significant differences in CD4+ or CD8+ cell infiltrates among the groups and differences in other cytokines were not identified by immunocytochemistry. Protection from clinical EAE in LR and co-immunized mice was partially abolished by anti-TGF-beta antibody treatment. Thus, protection from clinical disease following immunization with the analog LR is associated with infiltration into the CNS of a T cell population that could potentially recognize the native PLP peptide and with enhanced TGF-beta production by cells within CNS inflammatory foci.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease inducible in susceptible animals by myelin Ag-specific CD4+ Th1 cells. The mechanisms by which these cells induce inflammation and demyelination in the central nervous system (CNS) are incompletely understood. To determine the roles of Fas and FasL in the involvement of CNS autoimmune injury, we determined susceptibility to EAE of Fas-or FasL-deficient mice. Compared with wild-type mice, mice expressing lpr (Fas) and gld (FasL) mutations were relatively resistant to the development of clinical EAE, and this correlated with fewer inflammatory infiltrates and cells undergoing apoptosis in the CNS of the mutant mice. The gld and lpr mice, however, developed significant T cell responses with production of Th1 cytokines in response to the encephalitogenic myelin peptide. These results suggest that the Fas/FasL pathway plays a critical role in the development of EAE probably by mediating apoptosis within the target tissue.

Abstract

We employed factors analysis to quantify the degree of histologic heterogeneity of childhood infratentorial neuroglial tumors. Our data were 26 reliably ascertained histologic features in 1068 children in the Childhood Brain Tumor Consortium database. The factor analysis identified five uncorrelated quantitative "factors," each derived from a different linear combination of the 26 histologic features, that accounted for much of the histologic variation. Histologic features differed in their importance in each factor. The most important features in each factor were used for naming using simple, histologic, familiar descriptive terms: Spongy, Proliferative, Ring, Fibrillary, and Nuclear. Each tumor has a score on each factor. Two-thirds of tumors had high scores for at least two factors, indicating frequent histologic heterogeneity among these tumors. Ninety-five percent of tumors were allocated to 1 of 11 nonoverlapping histologically homogeneous groups. The five quantitative factors complement standard qualitative taxonomies by making explicit the histologic heterogeneity or homogeneity of individual tumors and provide the pathologist with a method that takes advantage of more of the histology of each tumor than conventional nomenclatures. Histologically homogeneous groups of tumors are likely to be of value in clinical trials and biologic research. Prognostic models based on these factors have been published.

Abstract

Myelin proteolipid protein (PLP) contains 2 immunodominant encephalitogenic epitopes in SJL mice, namely PLP residues 139-151 and 178-191. DM20, a minor isoform of PLP, lacks residues 116-150 and consequently contains only the single major encephalitogenic epitope 178-191. However, it has been found previously that bovine DM20 is not encephalitogenic in SJL mice. Since residue 188 within peptide 178-191 is phenylalanine (F) in murine DM20 and alanine (A) in bovine DM20, we tested the effect of this difference on the immune responses and induction of EAE. SJL mice were immunized with either highly purified murine or bovine DM20. Residues 178-191 were found to be immunodominant for each, but only murine and not bovine DM20 was encephalitogenic. A synthetic peptide corresponding to the murine 178-191 sequence (F188) was also encephalitogenic, whereas the peptide corresponding to the bovine sequence (A188) was not. Both F188 and A188 bind with high affinity to I-As and both are recognized by the SJL T cell repertoire. A188-specific T cell lines reacted to both A188 and F188, but F188-specific T cell lines were not stimulated by A188. F188-specific T cell lines produced mRNA for the Th1 cytokines IL2 and IFN gamma and, in passive transfer experiments, were encephalitogenic upon stimulation with F188, but not A188. In contrast, A188-specific T cell lines produced mRNA for IL4, IL5 and Il10, in addition to IL2 and IFN gamma, and were not encephalitogenic after stimulation with either F188 or A188. Cotransfer of A188-specific T cell lines with F188-specific T cell lines resulted in protection from EAE. Thus, A188 induces a functionally different phenotype of T cells from that induced by F188. Taken together these data suggest that the failure of bovine DM20 to induce EAE may be attributable to induction of protective rather than pathogenic T cell by the immunodominant epitope.

Abstract

A premature male baby (28 weeks gestational age) was delivered by Cesarean section and required ventilation for respiratory distress syndrome during the first postnatal week. Four weeks postnatally, he had an episode of transient renal failure followed by lethargy leading to coma. Ultrasound changes were interpreted as intraventricular hemorrhage, grade 2. The baby died 31 days after birth. Autopsy showed bilateral thrombosis of the deep cerebral veins.

Abstract

Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, prevented the clinical development of experimental autoimmune encephalomyelitis (EAE) with a reduction in inflammation and demyelination. Administration of AG reduced the expression of nitrosotyrosine in inflammatory lesions in the central nervous system. Cytokine expression, determined by semiquantitative PCR, revealed increased expression of IFN-gamma, IL-10, and TGF-beta, which was associated with protection from EAE, and reduced TNF-alpha, associated with the development of EAE. Furthermore, AG blocked the secretion of nitric oxide, TNF-alpha, and PGE2 in astrocyte cultures. AG did not influence the proliferation response of T cells to a pathogenic epitope of myelin basic protein. Down-regulation of nitric oxide by AG has widespread consequences for cytokine production in central nervous system inflammation and prevents EAE.

Abstract

In addition to an antigen-specific signal, T cell activation requires an antigen-independent costimulatory signal provided by interaction of CD28 with B7 (CD80 and CD86) on the APC. By blocking B7 interactions, previous studies demonstrated the requirement for costimulation in the induction of experimental allergic encephalomyelitis (EAE). Recent studies suggest that unlike CD28, CTLA-4 (a second B7 ligand) delivers an inhibitory signal. To address the regulatory role of CTLA-4 in EAE, we used an antibody directed against CTLA-4 administered at the time of disease induction. This resulted in a significantly more severe clinical course and more inflammatory and demyelinating lesions in the CNS of anti-CTLA-4-treated mice. These data suggest that CTLA-4-mediated inhibitory signals can regulate the clinical severity and histologic parameters of neuroautoimmune disease.

Abstract

Experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis, is a T cell-mediated autoimmune disease that can be induced in experimental animals by immunization with myelin Ags. Inbred strains of mice show varying degrees of susceptibility to EAE, indicating that susceptibility is an inherited trait. To define the genetic factors that control susceptibility to EAE, we performed linkage analysis on the first backcross (BC1) between highly susceptible SJL/J mice and resistant B10.S mice, both of which are of the H-2s haplotype. Mice were immunized for disease with encephalitogenic myelin proteolipid protein peptide 139 to 151, and analysis was performed on 68 backcross mice showing the severe disease phenotype (disease score > or = 3)and 68 backcross mice of the resistant phenotype (no clinical or histologic signs of disease) using microsatellite markers covering >98% of the genome. We found the strongest linkage (p = 0.001) with clinical disease at two loci: one at the telomeric end of chromosome 2, and another near the center of chromosome 3. In addition, several other regions showing some evidence of linkage (p < or = 0.05) with clinical disease were found.

Abstract

Antigen-driven tolerance is an effective method of suppressing cell-mediated immune responses. We have previously demonstrated that exposure of gut-associated lymphoid tissue to myelin basic protein (MBP) via oral administration suppresses experimental autoimmune encephalomyelitis (EAE). To further study presentation of antigen to the immune system by mucosal surfaces as a method of antigen-driven tolerance, the effect of inhalation of MBP was investigated. MBP was given as an aerosol to Lewis rats on Days -10, -7, -5, and -3 prior to immunization with MBP in Freund's adjuvant and on Days 0, 2, and 4 following immunization. Aerosolization of MBP completely abrogated clinical EAE in 100% of treated rats. Central nervous system inflammation and delayed-type hypersensitivity and antibody responses to MBP were also significantly reduced in aerosol-treated animals. Aerosolization of histone, a basic protein of similar weight and charge as MBP, had no effect. Disease was also suppressed with one aerosol treatment on Day -3 or by administering MBP nasally. Aerosolization was more effective than oral administration of MBP over a wide dose range (0.005-5 mg). Splenic T cells isolated from animals postaerosolization adoptively transferred protection to naive animals immunized with MBP. Aerosolization of MBP to animals with relapsing EAE after recovery from the first attack decreased the severity of a subsequent attack. Aerosol and oral MBP were equally effective at suppressing the in vitro immune response as measured by proliferation and interferon-gamma production. We then tested aerosolization of a different autoantigen in a different disease model and found that aerosolization of type II collagen was effective in suppressing collagen-induced arthritis. Thus, aerosolization of an autoantigen is a potent method to downregulate an experimental T cell-mediated autoimmune disease and suggests that exposure of antigen to lung mucosal surfaces preferentially generates immunologic tolerance.

Abstract

Semliki Forest Virus (SFV) causes a more severe acute encephalomyelitis in B6 than in SJL mice despite similar T cell proliferation and antibody responses in these two strains. To determine the immunological mechanisms that may contribute to this difference, CNS tissues from SFV-infected B6 and SJL mice were analyzed for viral replication, inflammatory responses and cytokine production, by semiquantitative reverse transcriptase-PCR and immunohistochemistry. Although initially similar on day 2 p.i., SFV replicated to higher viral titers in B6 than SJL mice on days 4 and 7 p.i. Infectious virus was cleared from both strains by day 10 p.i. There were no differences in numbers of CD4+, CD8+ or MHC class I and II+ inflammatory cells at any time point. Higher levels of IL-4 mRNA, lower levels of TNF-alpha, IL-6, IL-1 beta and IL-2 mRNAs and lower IL-2+ and IFN-gamma+ cells were found in B6. These findings suggest that despite comparable immune responses, different patterns of cytokine production correlated with higher levels of virus in the brains and more severe clinical disease in B6, and more efficient clearance of virus and less severe disease in SJL mice.

Abstract

Matrix metalloproteinases (MMPs) comprise a family of proteolytic enzymes that are involved in remodeling of the extracellular matrix (ECM) of many tissues. They have been implicated in degradation of vascular basement membranes thereby facilitating leukocyte migration into inflammatory sites. To determine the cellular localization and levels of MMPs in the normal human central nervous system (CNS), multiple sclerosis (MS) lesions, and other conditions, cryostat sections of CNS samples were immunostained with antisera to MMP-1, -2, -3 and -9. In control white matter the principal cells that express the MMPs were perivascular and parenchymal microglia. Cellular MMP expression was also found in sporadic microglial nodules in MS white matter. Most CNS microvessel endothelial cells expressed MMP-3 and -9 but not MMP-1 or -2. The majority of macrophages in active MS and necrotic lesions were MMP-l-, -2-, -3-, and -9-positive whereas chronic MS lesions had fewer MMP-positive macrophages. Small numbers of astrocytes were MMP-2-, -3- and -9-positive in acute and chronic MS lesions. These data suggest that microglia-derived MMPs may mediate turnover of the CNS ECM under normal conditions and in microglial nodules. In sites of CNS tissue injury there is complex and dynamic regulation of MMP expression by different cell populations. In MS lesions MMP-mediated proteolysis may contribute to breakdown of the blood-brain barrier and leukocyte migration into the CNS, in situ immune activation, demyelination, metabolism of bioactive peptides, and the formation of an ECM that does not promote remyelination or axonal repair.

Abstract

In experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, we have previously shown that the disease is mediated by Th1 cells, which recognize tryptophan 144 as the primary TCR contact point. Here we describe an altered peptide ligand (APL), generated by a single amino acid substitution (tryptophan to glutamine) at position 144 (Q144), which inhibits the development of EAE induced with the native PLP 139-151 peptide (W144). We show that the APL induces T cells that are cross-reactive with the native peptide and that these cells produce Th2 (IL-4 and IL-10) and Th0 (IFN gamma and IL-10) cytokines. Adoptive transfer of T cell lines generated with the APL confer protection from EAE. These data show that changing a single amino acid in an antigenic peptide can significantly influence T cell differentiation and suggest that immune deviation may be one of the mechanisms by which APLs can inhibit an autoimmune disease.

Abstract

CD4 T helper precursor cells mature along two alternative pathways, Th1 and Th2. Here we show that these pathways are differentially activated by two costimulatory molecules, B7-1 and B7-2. Using anti-B7 antibodies, this developmental step was manipulated both in vitro and in vivo in experimental allergic encephalomyelitis (EAE). Anti-B7-1 reduced the incidence of disease while anti-B7-2 increased disease severity. Neither antibody affected overall T cell induction but rather altered cytokine profile. Administration of anti-B7-1 at immunization resulted in predominant generation of Th2 clones whose transfer both prevented induction of EAE and abrogated established disease. Since co-treatment with anti-IL-4 antibody prevented disease amelioration, costimulatory molecules may directly affect initial cytokine secretion. Thus, interaction of B7-1 and B7-2 with shared counterreceptors CD28 and CTLA-4 results in very different outcomes in clinical disease by influencing commitment of precursors to a Th1 or Th2 lineage.

Abstract

Vitronectin (Vn) is a multifunctional plasma and extracellular matrix glycoprotein involved in cell attachment, coagulation, phagocytosis, and the protection of bystander cells from complement- and T cell-mediated lysis. To determine where Vn is localized and where cells expressing integrin Vn receptors may recognize it in central nervous system (CNS) lesions of multiple sclerosis (MS), CNS tissue samples were immunostained for Vn and the alphav, beta 1, and beta 3 integrin Vn receptor subunits. By light and electron microscopy, Vn was localized within dystrophic, demyelinated axons in active but not chronic lesions, normal or other neurologic disease controls. This localization is distinct from that of other plasma proteins in MS lesions and it differs from the pattern of neuron cell body localization found in other conditions. Microvascular Vn was increased and small numbers of reactive astrocytes were also Vn-positive in active plaques. Endothelial cell expression of the alpha v subunit was increased over controls and that of the beta 1 subunit was decreased whereas both the alpha v and beta 1 subunits were prominently expressed on macrophages and glia in active lesions. The beta 3 integrin subunit was expressed on platelets within and around vessels and was more prominent on endothelial cells in active plaques. The precise functions of Vn in situ are not presently known. These results indicate, however, that the regulation of expression of integrin Vn receptors is complex and that Vn may be recognized and have multiple functions in different microanatomic sites as MS lesions evolve. Intravascular Vn could participate in clotting, thereby contributing to leukocyte extravasation.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract

Multiple sclerosis (MS) is a chronic neurologic disease characterized in early phases by a cellular immune response and later by multiple areas of demyelination or plaques in the central nervous system (CNS) white matter. The clinical manifestations of the disease are highly variable, but probably are related to the extent of breakdown of the blood-brain barrier associated with inflammation in the acute phase, and with the extent of demyelination in the chronic phase. The initiating events of MS are not known, but current hypotheses include immune responses to an initiating viral infection and autoimmune responses to CNS myelin antigens. Both inflammatory and CNS resident cells contribute to the immunopathology of the disease. Chronic lesions are characterized by glial scarring and depletion of both oligodendrocytes and axons.

Abstract

Previously, six T cell clones, which are specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) peptide residues 139 to 151 (HSLGKWLGHPDKF), were derived from SJL mice and shown to use diverse TCR genes. To design TCR antagonist peptides that could interfere with the activation of these clones in vitro and inhibit experimental allergic encephalomyelitis (EAE) in vivo, we first determined the TCR and MHC contact residues of the encephalitogenic peptide. The analysis indicated that residues 144 (tryptophan) and 147 (histidine) were the TCR binding sites and that residues 145 (leucine) and 148 (proline) were important for MHC class II (IAs) binding. On the basis of this information, a peptide analogue (leucine 144/arginine 147), in which both of the major TCR contact residues were substituted, was synthesized. This analogue acts as a TCR antagonist for the panel of PLP 139-151-specific T cell clones, does not cause EAE by itself, blocks the induction of disease by the native 139-151 peptide, and prevents clinical disease progression if administered at the first signs of disease. Thus, although multiple TCR genes are used by PLP 139-151-specific clones, a single peptide analogue can interfere with the disease process. This approach should be feasible for designing peptide analogues that can be tested for therapeutic efficacy in human autoimmune diseases in which the pathogenic Ags are known and TCR use is diverse.

Abstract

The authors present a brief historical sketch of the development of our understanding of immune responses to myelin proteolipid protein (PLP) and the acceptance of PLP as a potent antigen in the induction of experimental allergic encephalomyelitis (EAE). The distinct characteristics of the PLP molecule that may contribute to complex immune responses to this protein are reviewed and these responses are compared with those to MBP, both in the pathology of EAE and at the level of the T cell. Recent evidence demonstrating differences between T cell responses to PLP and MBP is reviewed. Finally, the potential contribution of immune responses to PLP in human diseases, particularly multiple sclerosis (MS), that have been identified to date are then summarized.

Abstract

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease that can be induced in laboratory animals by immunization with the major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the role of the T cell receptor (TCR) repertoire in susceptibility to EAE induced by these two autoantigens. Autoreactive T cells induced after immunization with MBP use a limited set of TCR. In contrast, we demonstrate that T cell clones that recognize the encephalitogenic PLP epitope (PLP 139-151) use diverse TCR genes. When the TCR repertoire is limited by introduction of a novel rearranged TCR V beta 8.2 chain in transgenic SJL mice, EAE could be induced in the transgenic mice by immunization with the encephalitogenic epitopes of PLP, but not with the encephalitogenic epitope of MBP. Thus, skewing the TCR repertoire affects the susceptibility to EAE by immunization with MBP but not with PLP. These data demonstrate the biological consequences of the usage of a more diverse T cell repertoire in the development of an autoimmune disease.

Abstract

Determination of the topographic orientation of proteolipid protein (PLP) within myelin is part of an overall understanding of the functions of PLP and the roles of its multiple domains in diseases that primarily affect central nervous system (CNS) myelin. As part of an analysis of PLP orientation, two mouse monoclonal antibodies (mAb) and a rabbit antiserum against a synthetic peptide corresponding to PLP residues 103-116 (YKTTICGKGLSATV) were tested for their reactivity on compact CNS myelin. By ELISA, the antibodies react with intact PLP and PLP residues 103-116, but not with other PLP peptides. Ultrathin cryosections of adult rat optic nerve were immunostained and antibody binding was localized using appropriate second antibodies coupled to 1 nm gold particles that were visualized by silver enhancement. Localization of the particles on the major or intermediate dense lines was determined by three independent observers. Using the PLP peptide mAb and the polyclonal antibody, we demonstrated that > or = 71% of the particles were localized on the major dense line. At least 66% of particles directed against myelin basic protein, which is known to occur on the major dense line, were also found in that location. These semiquantitative morphologic observations suggest that PLP residues 103-116 occur on the cytoplasmic face of the myelin membrane.

Abstract

The histologic distinctions between normal choroid plexus and choroid plexus papilloma and between choroid plexus papilloma and choroid plexus carcinoma are sometimes difficult. The authors performed the silver nucleolar organizer region (AgNOR) technique, immunohistochemistry for proliferating cell nuclear antigen (PCNA), and DNA ploidy analysis by flow cytometry on 9 samples of normal choroid plexus, 8 papillomas, and 13 carcinomas to evaluate whether these techniques can aid in these differential diagnoses. Significant differences were found in the mean AgNOR count between normal choroid plexus (1.35 +/- 0.11) and choroid plexus papillomas (2.42 +/- 0.81) (P < 0.001), but not between choroid plexus papillomas and carcinomas. In the normal choroid plexus, AgNORs were smooth and round; in the papillomas and carcinomas, however, they varied in size and shape. Compound AgNORs were commonly present in the tumors but were essentially absent in controls. Antibody to PCNA did not stain normal choroid plexus cells (except for focal staining in one sample of normal choroid plexus adjacent to a carcinoma) but stained many papilloma and carcinoma cells. DNA ploidy analysis demonstrated aneuploidy in some papillomas and carcinomas but could not be used for the distinction of normal choroid plexus from papillomas. These results suggested that the AgNOR technique and PCNA immunohistochemistry could be used to distinguish normal choroid plexus from choroid plexus papilloma in small, diagnostically difficult biopsy specimens.

Abstract

We have derived a panel of CD4+, TCR-alpha/beta + T cell clones from SJL (H-2s) mice specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) 139-151 (HSLGKWLGHPDKF). All the clones are Ag specific and IAs restricted, but they show heterogeneity in their ability to induce experimental allergic encephalomyelitis (EAE), i.e., one group induces EAE in naive mice, a second group induces disease only in mice that are pretreated with pertussis and irradiation, whereas a third group is essentially nonencephalitogenic. To determine the basis for this functional heterogeneity, the clones were tested for the expression of adhesion molecules and cytokines and for Ag-specific cytolytic activity. All of the clones expressed comparable levels of LFA-1 and CD44 but lacked expression of Mel 14. However, those clones that induced EAE only in irradiation- and pertussis-treated recipients did not express VLA4. Because pretreatment with pertussis has been suggested to increase permeability of the blood-brain barrier and facilitate migration of T cells into the central nervous system, the absence of VLA4 on this group of clones may account for the need for pretreatment to induce EAE. The nonencephalitogenic clones expressed all of the adhesion molecules tested but were not cytolytic in vitro and failed to produce one or more of the proinflammatory cytokines after Ag-specific stimulation. One nonencephalitogenic clone that did not produce many cytokines on activation with specific Ag, however, could be activated with Con A to express mRNA for most cytokines and this was accompanied by a concomitant change in the encephalitogenic potency of this clone. These results suggest that adhesion molecules and cytokines both play a critical role in the encephalitogenicity of PLP peptide-specific T cell clones. Furthermore, the nonencephalitogenicity of some clones may be related to a defect in Ag-mediated activation.

Abstract

Antigen-driven tolerance is an effective method of suppressing cell-mediated immune responses. We have previously shown that oral administration of myelin basic protein (MBP) suppresses experimental autoimmune encephalomyelitis (EAE) when it is actively induced by MBP emulsified in complete Freund's adjuvant. In order to further study antigen-driven tolerance in this model, we investigated the effect of oral tolerization on adoptively transferred EAE and compared oral tolerance to intravenously (i.v.) administered MBP in both actively induced EAE and adoptively transferred EAE. Although orally tolerized animals were not protected from adoptively transferred EAE, spleen cells from orally tolerized animals suppressed adoptively transferred EAE when co-transferred with encephalitogenic cells or when injected into recipient animals at a different site at the time encephalitogenic cells were transferred. This suppression was mediated by CD8+ T cells, correlated with suppression of DTH responses to MBP, and was associated with decreased inflammation in the spinal cord. Unlike oral tolerization, spleen cells from i.v. tolerized animals did not suppress adoptively transferred EAE when co-transferred with encephalitogenic cells although i.v. tolerized animals were protected from adoptively transferred EAE. MBP peptides were then utilized to further characterize differences between i.v. and oral tolerization in the actively induced disease model. Both orally and intravenously administered MBP suppressed actively induced EAE. However, EAE was only suppressed by prior i.v. tolerization with the encephalitogenic MBP peptide 71-90, but not with the non-encephalitogenic peptide 21-40, whereas prior tolerization with 21-40 did suppress actively induced EAE when administered orally. These results suggest a different mechanism of tolerance is initiated by oral vs. intravenous administered antigen. Specifically, oral tolerization suppresses primarily by the generation of active suppression whereas the dominant mechanism of suppression associated with i.v. tolerization appears most consistent with the elicitation of clonal anergy.

Abstract

In strains of mice that are susceptible to experimental autoimmune encephalomyelitis (EAE), cloned CD4+ T cells reactive with autologous myelin basic protein (MBP) have been shown to cause disease when transferred to naive syngeneic recipients. Recent reports indicate that under particular experimental conditions, 'resistant' strains of mice can also develop EAE, although cloned cells have not been isolated and characterized. An analysis of the characteristics of a panel of MBP-specific T cells and the antigen presenting capability of CNS-derived cells obtained from the resistant strain BALB/c is presented here. The data demonstrate that immunization of EAE-resistant BALB/c mice results in the activation of a heterogeneous group of T cells reactive with autologous MBP. Both peripheral antigen presenting cells, as well as microglia isolated from brains of BALB/c mice, are capable of stimulating these cloned MBP-specific T cells to proliferate. When optimally activated in vitro and then injected in vivo into syngeneic BALB/c recipients, three clones studied induced severe cachexia, resulting in loss of up to 35% of body weight before death. Two of the clones also induced clinical and histological EAE, while the third induced only occasional histological evidence of disease. Differences in epitope recognition, T cell receptor usage, cytokine profiles or regulatory mechanisms of self tolerance, may play important roles in preventing potentially destructive autoimmune reactions by these T cells capable of recognizing autologous myelin in the central nervous system.

Abstract

The repertoire of TCR V beta genes transcribed and expressed within the central nervous system was determined in mice with experimental allergic encephalomyelitis. Disease was induced in (PL/J x SJL/)F1 mice by immunizing with myelin basic protein-acetylated peptide 1-11, and mice were sacrificed at intervals from day 3 postimmunization to 3 wk after recovery from disease. Transcription of V beta genes was determined by reverse transcriptase polymerase chain reaction on RNA extracted from spinal cord, and expression of the V beta gene products was detected by immunohistochemistry with mAb specific for various V beta proteins. Multiple V beta genes were found to be transcribed and expressed in the central nervous system starting 7 days after immunization, and continuing up to 3 wk after clinical recovery. Preferential utilization of a single TCR V beta gene was not detected in the central nervous system at any time in the course of disease.

Abstract

Unilateral vestibular schwannomas (VS) differ from those in patients with neurofibromatosis 2 (NF-2) clinically and by in situ appearance. To determine whether there are histopathologic differences, the presence of each of 16 histologic features was compared in first surgical resection specimens of 48 VS from 39 NF-2 patients and 293 unilateral VS. Antoni A and B areas, nuclear atypia, whorls, scarring, chronic inflammation, and sheets of macrophages were found equally in both groups. Vestibular schwannomas in NF-2 had more Verocay bodies, foci of high cellularity, and lobular growth patterns, the latter possibly correlating with in situ appearance. Ten NF-2 VS specimens had either meningiomas or microscopic meningeal cell proliferations removed with the VS from the same area, whereas none of the patients with a unilateral VS had these findings. Unilateral VS had more hyalinized and malformed vessels, recent and old thromboses and hemosiderin deposits. The differences could not be attributed to patient ages because there were similar differences between the VS in NF-2 and the unilateral VS of 40 patients age-matched to the NF-2 patients. There were more female patients in both groups, but gender did not influence the occurrence of any histologic features. There were nine additional patients with apparently unilateral VS but in whom the diagnosis of NF-2 was suggested by additional findings; six of the VS from these patients had lobular patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract

To develop a reproducible in vivo model for the growth of human acoustic neuromas and neurofibromas, we implanted tumor specimens (6 acoustic neuromas; 4 neurofibromas; 3 schwannomas arising in skin and soft tissues) from 13 different patients into the subrenal capsules of 67 nude mice and sciatic nerves of 64 nude mide. The animals were anesthetized and the tumors were microscopically implanted. Serial tumor volumes were determined at intervals up to 2 months by reopening the incision and directly measuring the tumor size with a micrometer. The percentages of acoustic neuromas that survived or grew were 57.1% in the subrenal capsule and 88.9% in the sciatic nerves; the percentages of neurofibromas that survived and grew were 50% in the subrenal capsule and 70% in the sciatic nerves; and the percentages of schwannomas that survived and grew were 57.1% in the subrenal capsule and 94.1% in the sciatic nerve. Tumors in the sciatic nerve also survived and grew for a longer period than those in the subrenal capsules. Tumor enlargement and stability correlated with neovascularity. At 1 or 2 months after engraftment, the tumors showed histologic appearances similar to the original tumors and immunohistochemical analysis of cryostat sections demonstrated staining of the tumors, but not the host mouse tissues for human beta 2-microglobulin, a species-specific marker. Furthermore, analysis of genomic DNA from implanted tumors revealed its human origin. We conclude that human acoustic neuromas, neurofibromas and schwannomas are readily grown in two sites in nude mice and that they retain their morphologic features and genomic identities. These tumors grow better and more consistently in the sciatic nerve than in the subrenal capsule. These are useful model systems for studying tumor growth and cellular modulation.

Abstract

Children whose brain tumor involves two or more compartments at presentation differ clinically and pathologically from children whose brain tumor is confined to one compartment. In this study of 3,291 children with a brain tumor, at least 10% had a tumor that occupied two or three compartments at first hospitalization. Infratentorial tumors occupying multiple compartments were 1.7 times more likely to involve the cervicomedullary junction than the mesodiencephalic junction. Younger children (1-3 years) were more likely to have had multiple compartment tumors than older children. Children whose tumor was limited to the infratentorial compartment had a longer survival than children whose tumor also occupied other compartments. Ependymoma, anaplastic ependymoma, and astrocytoma (nos) were over represented among infratentorial multiple compartment tumors. Pilocytic astrocytoma, primitive neuroectodermal tumor (medulloblastoma), and desmoplastic medulloblastoma were less likely to have occupied multiple compartments at the time of the first surgical exploration. The distributions of histologic features in tumors at the cervicomedullary junction differed from those in tumors limited to the posterior fossa or to the spinal canal. Seizures were more likely if the tumor was confined to the supratentorial compartment, whereas nausea or vomiting and headache were more likely if the tumor was confined to the infratentorial compartment. Children whose tumor was confined to the spinal canal were significantly more likely to have bladder symptoms and back and/or abdominal pain than those whose tumor also involved compartments above the foramen magnum. We conclude that brain tumors apparently confined to one compartment at presentation are biologically and structurally different from tumors evident in two or more compartments.

Abstract

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.

Abstract

We previously described a synthetic peptide of myelin proteolipid protein (PLP), peptide 139-151, which induces experimental allergic encephalomyelitis in SJL/J (H-2s) mice. We have now identified an additional determinant, PLP residues 178-191, that is also a potent encephalitogen in this strain. When PLP peptide 178-191 was compared with peptide 139-151 on an equimolar basis, the day of onset of disease induced by PLP 178-191 was earlier, but the incidence, severity, and histologic features were indistinguishable. Lymph node cells from animals immunized with the whole PLP molecule responded to both PLP 178-191 and 139-151, suggesting immunologic codominance of the two epitopes. PLP 178-191 elicited stronger proliferative responses and this may relate to the earlier onset of disease induced with this peptide. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice. The presence of multiple encephalitogenic codominant PLP epitopes within a single mouse strain emphasizes the complexity of the immune response to PLP and its potential as a target Ag in autoimmune demyelinating diseases.

Abstract

Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in SWR/J (H-2q) and SJL/J (H-2s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In SWR/J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in SWR/J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.

Abstract

Proteolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 develop acute EAE. In this study, 6 IAs-restricted, CD4+, TCR alpha beta-bearing T cell clones were derived from SJL/J mice after immunization with this synthetic peptide. The clones responded in in vitro proliferative assays to the whole PLP molecule and to PLP peptide 139-151, but not to irrelevant Ag. They also responded to truncated and overlapping forms of the peptide but five distinct reactivity patterns were observed using these peptides. A panel of anti-TCR V beta mAb and TCR V beta-specific cDNA probes were used to determine the TCR V beta usage of the clones. Five clones were found to use four different V beta (V beta 2, V beta 6, V beta 10, or V beta 17a), whereas the V beta on the sixth clone could not be identified. Five of the clones induced EAE of varying severity upon adoptive transfer into naive syngeneic mice or mice pretreated with irradiation and pertussis and one clone was nonencephalitogenic. The Ag-specific proliferative response of all but the nonencephalitogenic clone could be blocked by an anti-CD4 mAb. Thus, the clones showed differences in their fine specifity, TCR V beta usage, sensitivity to antibody blocking, and encephalitogenic potency. These data demonstrate that the T cell response to the encephalitogenic PLP peptide 139-151 is heterogeneous.

Abstract

We sought temporal trends in the demographic, clinical, histologic feature, diagnostic class, and quality of life data over the interval 1930-1979 in the Childhood Brain Tumor Consortium database. The proportion of children younger than eight years old declined from 72% to 55% and the proportion of those older than ten more than doubled from 12% to 27%. The relative frequency of tumors in the supratentorial compartment increased significantly, while infratentorial tumors decreased. We found significant declines in supratentorial ependymomas and pilocytic astrocytomas. Similarly, some infratentorial tumors, especially ependymomas, decreased and brain stem tumors increased. Infratentorial medulloblastoma (primitive neuroectodermal tumor) increased significantly. Some individual histologic features which are markers of anaplasia increased in frequency in both supratentorial and infratentorial tumors. There was a significant increase in biopsies that contained nonneoplastic neural tissue in addition to tumor for both compartments and among supratentorial tumors there was a marked increase in the proportion of cases containing an indistinct neural tissue boundary. The probability of postoperative death declined, but the probability of survival five or ten years after surgery did not improve significantly for children who had tumors in either compartment. Among children who survived five years after the initial craniotomy, the proportion who had significant long term deficits increased. Most of this increase occurred in the last decade (1970-79). In this decade, the proportion of children for whom no deficits were reported five years following operation was 4% if they had a supratentorial tumor and 27% if they had an infratentorial tumor. The proportions of children alive five years following first surgery who had arachnoidal metastases increased significantly for infratentorial tumors.

Abstract

We examined potential clinical and pathologic correlates of seizures among the 3,291 children in the Childhood Brain Tumor Consortium database. Fourteen percent had seizures prior to their hospitalization for a brain tumor. Among children who had a supratentorial tumor, seizures occurred in 22% of those less than 14 years of age. The prevalence of seizures increased to 68% of older teenagers. Among children with an infratentorial tumor, the prevalence of seizures was relatively constant at 6% over all age groups. The onset of seizures began more than one year prior to surgical tumor removal in over half of the children aged five or more with supratentorial tumors, significantly longer than for those of the same age with infratentorial tumors. Almost all children (98.9%) with an infratentorial tumor and seizures had at least one other symptom and more than three-fourths of them had at least three. Eighty-nine percent of children with a supratentorial tumor and seizures had at least one other symptom and more than one-half had at least three symptoms. Regardless of whether the tumor was above or below the tentorium, confusion or stupor and coma were more common in children with seizures than in children without seizures. Among children with supratentorial tumors, symptoms of a declining academic performance or an abnormality of personality, speech, walking, or sensation were significantly more frequent in children with seizures, while visual symptoms (other than visual loss or diplopia) and nausea or vomiting were less frequent. Among children with supratentorial tumors, those who had seizures were more likely to have paralysis of an arm, hand, or face, confusion or stupor, or coma and less likely to exhibit irritability, papilledema, optic atrophy, decreased visual acuity, pupillary abnormalities, or abducens paresis. Among children with infratentorial tumors, those with seizures were significantly less likely to have truncal ataxia, but more likely to experience confusion, stupor, or coma. In the supratentorial compartment, astrocytoma (nos), protoplasmic astrocytoma, anaplastic astrocytoma, and ependymoma were more frequently associated with seizures than was craniopharyngioma. No infratentorial tumor type was more or less likely to be associated with seizures. All common tumor types that were represented in both the supratentorial and the infratentorial compartment except astrocytoma (nos) were associated with significantly greater rates of seizures when located in the supratentorial compartment. The tumor location with the highest incidence of seizures was, as expected, the superficial cerebrum. More than 40% of the children with such tumors had seizures.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract

Proteolipid protein (PLP) is the major protein of central nervous system (CNS) myelin. SJL(H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 (HSLGKWLGHPDKF) develop acute experimental allergic encephalomyelitis (EAE). In the present study a T cell line and 4 clones were derived from SJL/J mice following immunization with this synthetic peptide. Severe clinical and histological EAE could be induced by adoptive transfer of the peptide-specific T cell line and 3 of 4 T cell clones. The T cell line/clones all responded strongly to PLP peptide 139-151 in in vitro proliferative assays. However, two different reactivity patterns emerged when truncated PLP peptides 141-150 and 141-149 were tested, suggesting that more than 1 epitope may be present within the PLP 139-151 determinant. To evaluate the encephalitogenic potential of the truncated peptides, we compared the ability of 2 truncated PLP peptides to induce EAE in vivo and proliferative responses in vitro. Immunization with PLP peptide 141-150 induced acute EAE in about 70% of mice tested, but PLP peptide 141-149 induced a comparatively mild form of EAE in 4 out of 9 mice tested. Lymph node cells from mice immunized with these peptides showed in vitro proliferative responses to each of the peptides, but the response to peptide 139-151 was always strongest. These combined in vivo and in vitro data further define the epitopes involved in PLP-induced EAE in SJL mice. Furthermore, the availability of multiple PLP-specific T cell clones will enable us to study the diversity of the T cell repertoire to PLP.

Abstract

Oral administration of proteins is a long-recognized method of inducing antigen-specific peripheral immune tolerance. We previously showed that oral administration of myelin basic protein suppresses monophasic experimental autoimmune encephalomyelitis in the Lewis rat when it is given in association with immunization and prior to disease onset. As a potential therapy for human autoimmune disease, it is crucial to determine whether oral tolerance can ameliorate an ongoing immune response. We therefore asked whether oral administration of myelin antigens, after sensitization and disease expression has occurred, could affect immunological, clinical, or pathological features of experimental autoimmune encephalomyelitis. Chronic relapsing experimental autoimmune encephalomyelitis was induced in the Lewis rat and strain 13 guinea pig by immunization with whole guinea pig cord homogenate, complete Freund's adjuvant, and Mycobacterium tuberculosis. Following recovery from the first attack, animals were orally given bovine myelin, guinea pig myelin, or guinea pig myelin basic protein three times per week for up to 3 months. Animals receiving myelin products orally had decreased severity and frequency of clinical relapses, decreased delayed-type hypersensitivity responses to myelin antigens, diminished inflammation in the central nervous system (CNS), and decreased areas of CNS demyelination. In the rat, guinea pig myelin basic protein was as effective as guinea pig myelin in ameliorating the disease and also resulted in decreased serum anti-myelin basic protein antibody levels. No exacerbation of disease or worsening of pathological findings occurred in the animals given myelin products. These results demonstrate that oral administration of myelin antigens can suppress chronic relapsing experimental autoimmune encephalomyelitis and have direct relevance to therapy of human demyelinating disorders such as multiple sclerosis.

Abstract

The potential of multiparametric proton magnetic resonance (MR) measurements for characterizing white matter lesions was investigated. The authors compared acute experimental allergic encephalomyelitis (EAE), which is distinguished by inflammatory lesions, with an immunologically potentiated hyperacute form of the disease in which demyelinating lesions (DEM) also are present. Tissue samples containing cervical spinal cord and brain stem were excised and in vitro measurements of T1, T2, and two components of T2 were performed. Discriminant analysis was applied using MR parameters singly and in various combinations. When the disease was clearly manifested, discrimination between treated and normal animals was satisfactory with single parameters. The use of biexponential T2 components improved the distinction of normal from treated but asymptomatic animals, and differentiated between EAE and DEM. These results suggest that improved characterization of white matter lesions is possible with multiparametric MR in vivo, especially if sampling is performed with imaging and the T2 decay curves are obtained with a sufficient number of echoes to perform biexponential analysis.

Abstract

Benign spinal nerve sheath tumors (neurofibromas and schwannomas) often occur on dorsal nerve roots sporadically or in neurofibromatosis types 1 and 2. These are histologically benign tumors, and distinction between them is frequently not made by clinicians. To determine if there is a correlation between the histological pattern of benign spinal nerve sheath tumors and the type of neurofibromatosis, the clinical and pathological features of these tumors (86 surgical specimens and five autopsies) in 68 patients were reviewed. The patients were classified into one of four categories: neurofibromatosis type 1, neurofibromatosis type 2, uncertain, or sporadic. The diagnostic criteria used for neurofibromatosis types 1 and 2 were established by the National Institutes of Health. Patients who did not fulfill criteria for either neurofibromatosis type 1 or 2 but who had multiple nervous system tumors or other stigmata of neurofibromatosis were designated "uncertain." Spinal nerve sheath tumors were considered sporadic in 42 cases (40 schwannomas and two neurofibromas). In the 14 patients with neurofibromatosis type 1, all spinal nerve sheath tumors were neurofibromas. In six of the seven patients with neurofibromatosis type 2, all spinal nerve sheath tumors were schwannomas. One patient with neurofibromatosis type 2 had a spinal nerve sheath schwannoma and a tumor with features of both tumor types. The authors conclude that spinal nerve sheath tumors in patients with neurofibromatosis type 1 are neurofibromas. In contrast, spinal nerve sheath tumors occurring in neurofibromatosis type 2 or sporadically are most frequently schwannomas. The distinct histological features of these tumors may reflect different pathogenetic mechanisms even though they arise at identical sites in neurofibromatosis types 1 and 2.

Abstract

To determine if the Ag that induces an autoimmune disease influences parental MHC haplotype molecule expression in situ in MHC heterozygotes, acute experimental allergic encephalomyelitis (EAE) was induced with different encephalitogenic peptides in (SJL/J x SWR)F1 mice. The mice were sensitized with either a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 103-116 YKTTICGKGLSATV which induces EAE in SWR (H-2q), but not SJL/J (H-2s) mice or a synthetic peptide corresponding to PLP residues 139-151 HCLGKWLGHPDKF which is encephalitogenic in SJL/J but not SWR mice. Mice were killed when they were moribund or at 30 days after sensitization. Twelve of 18 F1 mice given PLP peptide 103-116 and 12 of 17 mice given PLP peptide 139-151 developed EAE within 2 to 3 wk after sensitization. Cryostat sections of brain samples from F1 and parental mice were immunostained with a panel of mAb identifying H-2s and H-2q class I and II MHC molecules. In brains of controls, class I MHC molecules were expressed on choroid plexus, endothelial cells, and microglia whereas class II MHC molecules were absent. In EAE lesions, class I and II MHC molecules were present on inflammatory and parenchymal cells, but the degree of parental haplotype molecule expression did not vary with the different peptide Ag tested. Thus, in (SJL/J x SWR)F1 mice, myelin PLP peptides 103-116 and 139-151 are co-dominant Ag with respect to clinical and histologic disease and parental haplotype MHC molecule expression. We propose a unifying hypothesis consistent with these results and previous observations of differential Ia expression in (responder x non-responder)F1 guinea pigs. We suggest that MHC molecules may bind locally derived peptide Ag in inflammatory sites and that these interactions influence levels of MHC haplotype molecules on APC.

Abstract

A sphenoid-wing meningioma in a 60-year-old woman was accompanied by elevated serum carcinoembryonic antigen (CEA) levels, which returned to normal after removal of the tumor. Light microscopic examination revealed a secretory meningioma containing numerous pseudopsammoma bodies and a prominent vascular pattern. Immunohistochemical analysis showed the tumor cells and pseudopsammoma bodies to be CEA-positive. This case illustrates the possibility that secretory meningioma may be associated with clinically detectable secretion of CEA. The report also documents the rare occurrence of elevated serum CEA in a primary benign intracranial tumor.

Abstract

A human neurofibrosarcoma was removed at surgery from a patient with neurofibromatosis and implanted into the subrenal capsule of female nude mice (nu/nu). A solid tumor grew and was transferred to 78 additional mice for this study. The animals were randomly assigned to one of four groups: 1) control, 27 animals; 2) oral heparin (200 or 500 U/ml), 17 animals; 3) oral hydrocortisone (0.3 mg/ml), 10 animals; or 4) oral heparin (200, 500, or 1000 U/ml) with hydrocortisone (0.3 mg/ml), 24 animals. After 10 days of treatment, the animals were sacrificed and the tumor size and degree of neovascularization were compared to the pretreatment data. Heparin treatment alone stimulated angiogenesis and resulted in tumor growth greater than in the control group (p less than 0.001). Administration of hydrocortisone alone caused a minimal reduction in tumor growth and had a minimal effect on angiogenesis (p less than 0.05 vs. control group). In contrast, heparin administered with hydrocortisone inhibited both angiogenesis and tumor growth (p less than 0.001 vs. control group). These studies suggest that angiogenesis modulators are worthy of further study as feasible means of treating human neurofibrosarcoma.

Abstract

Clinical, histologic, and ultrastructural characteristics of acute experimental allergic encephalomyelitis (EAE) induced by sensitization with a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 139-151 HCLGKWLGHPDKF were studied in SJL/J mice. Groups of mice were immunized with 20, 50, or 100 nmol of the peptide and were killed from seven to 28 days after sensitization or when they were moribund. Beginning on Day 9, the mice showed signs of EAE and the disease progressed rapidly to paralysis. Central nervous system (CNS) inflammation, edema, gliosis, and demyelination were found in all mice killed between Days 10 and 28 and white matter lesion areas correlated with clinical score at the time the mice were killed. Peripheral nerve roots and the cauda equina did not have lesions. Within the range studied, the severity of clinical or histologic disease was the same regardless of the PLP peptide dose. Two of ten mice immunized with 100 nmol and none of 14 mice given smaller doses of a synthetic peptide of mouse myelin basic protein (MBP) showed clinical EAE. These mice had small numbers of CNS lesions that were indistinguishable from those in PLP peptide-sensitized mice. These findings demonstrate that immunization of SJL/J mice with PLP peptide 139-151 produces a disease with the clinical and morphologic features of CNS tissue-, whole PLP-, whole MBP-, and MBP peptide-induced acute EAE. Thus, PLP is a major encephalitogen and immune reactions to epitopes of different myelin proteins may induce identical patterns of injury in the CNS.

Abstract

Monoclonal antibody immunocytochemistry was used to examine spinal cord and muscle in amyotrophic lateral sclerosis for changes that would indicate ongoing or potential immune activity. Increased expression of class I and II major histocompatibility complex (MHC) antigens was seen in the affected areas of spinal cord. New MHC expression was concentrated in phagocytes, particularly in degenerating white matter in which they were dispersed in the tissue and also packed around blood vessels. MHC antigen was not revealed in motor neurons or skeletal muscle fibers. An anti-pan-T-cell monoclonal revealed small numbers of T cells in degenerating white matter. Similar changes have been seen in other neurodegenerative disorders. They suggest a potential for (secondary) cell-mediated activity in the affected areas rather than an ongoing MHC-restricted T-cell response. Vessel-associated phagocytes may be a source of antigen to peripheral lymphoid tissue, stimulating production of the autoantibodies that have been described.

Abstract

Cryostat sections of human central nervous system (CNS) tissue samples from 10 cases of multiple sclerosis (MS), 11 cases with inflammation and necrosis, and 24 normal controls were immunostained with antibodies to intercellular adhesion molecule-1 (ICAM-1) and its integrin ligand lymphocyte function-associated molecule-1 (LFA-1). In 18 controls, small numbers of CNS microvessels were ICAM-1-positive. There were more numerous ICAM-1-positive vessels in active MS plaque edges, viral encephalitis lesions, infarcts, and in six controls. Within active MS plaques and in viral infections, mononuclear cells and some glia also were ICAM-1-positive. Mononuclear but not CNS resident cells were LFA-1-positive. Thus, CNS vessel ICAM-1 expression is variable in amount in postmortem samples of normal human CNS tissue, may increase early and focally in cellular immune reactions, and, via binding of LFA-1, may promote leukocyte influx into the CNS. Intercellular adhesion molecule-1 expression on parenchymal cells may indicate additional interactions with LFA-1 on inflammatory cells in diverse CNS lesions.

Abstract

To develop a reproducible in vivo model for the growth of human meningiomas, meningiomas from 16 patients were implanted into the subrenal capsule of the nude mouse. In eight experiments solid tumor implants taken directly from surgical specimens were used, in four experiments the implants were made from early-passage monolayer cell cultures, and in four experiments both techniques were used. Successful tumor growth was observed in 10 (83%) of the 12 solid tumor implants and in six (75%) of the eight implants from cell cultures. The size and neovascularization of these tumors were serially determined over a 3-month period. Tumor doubling occurred in 1 to 3 weeks in all of the solid tumor implant group. In the group of six tumors successfully implanted from cell cultures, three doubled in 1 to 3 weeks and three grew more rapidly, reaching 10 to 20 times their original volume. Neovascularity occurred in the tumors within 3 weeks of implantation. Each of the solid tumor implants had a histological pattern similar to that of the corresponding original specimen. Only three of those implanted from cell cultures were similar to the original tumor; the other three displayed features characteristic of malignant meningioma. These studies suggest that implantation of human meningiomas in the subrenal capsule of the nude mouse is a feasible model that may be useful for evaluating hormonal or genetic modulation of tumor growth and for testing potential treatment regimens.

Abstract

Cryostat sections of central nervous system (CNS) tissues of patients with multiple sclerosis (MS) and other CNS diseases were stained with antibodies to fibronectin, a macrophage fibronectin receptor component, fibrin/fibrinogen, and albumin using immunoperoxidase. In active, but not inactive, MS plaques vessel fibronectin was increased (to approximately 57% of Factor VIII+ vessels) over uninvolved MS and normal control white matter (P less than 0.001 for both). Fibronectin was primarily localized to vessel walls and amount of staining correlated with degree of inflammation. Active plaques and necrotic lesions also had extracellular fibronectin and fibrin/ogen. These molecules and the fibronectin receptor were found on macrophages. Albumin was more widely and diffusely distributed in lesions than fibronectin. Thus, in addition to extravasation from damaged vessels, fibronectin may be deposited on or synthesized by endothelial cells and macrophages in the CNS. Fibronectin could facilitate monocyte adhesion to endothelial cell luminal surfaces, promote migration of mononuclear cells, and enhance myelin phagocytosis in MS lesions.

Abstract

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.

Abstract

Ten human teratomas arising outside the central nervous system (CNS) were studied using a panel of immunohistochemical, and lectin histochemical stains to determine the relationship of the presence of microglia to markers of neural maturity or differentiation. Microglia, identified by silver carbonate, Ricinus communis agglutinin-1 (RCA-1), or both were found in eight out of ten cases. They were common in mature areas which also had S-100, glial fibrillary acidic protein, vimentin, neurofilament, and synaptophysin immunostaining. Microglia were distinguished from macrophages in necrotic foci. Cells which were RCA-1 positive and silver carbonate positive were found in immature neural tissues but these lacked all typical features of microglia. These observations indicate that true microglia are frequent in nonCNS teratomas and that they are found in association with other indicators of neural maturation. The presence of possible precursors in immature areas suggests that microglia undergo maturation concurrent with neural differentiation in these tumors.

Abstract

CNS demyelinating inflammatory disease can be a multifactorial process mediated by cellular and antibody-mediated immune processes. Myelin basic protein (MBP)-specific T cells and pathogenic 8-18C5 antibody, specific for a myelin/oligodendrocyte glycoprotein (MOG), a minor component of CNS white matter, can coexist in rats without triggering disease. However, transfer of activated MBP-specific T-cells followed by the injection of 8-18C5 antibody resulted in hyperacute disease progression and CNS demyelination. Transfer of activated T cells specific for an irrelevant antigen or transfer of activated but irradiated encephalitogenic T cells did not induce disease in the presence of 8-18C5 antibody. When needle lesions were induced in brains of 8-18C5 antibody treated rats, no enhancement of demyelination was seen around the needle track. Thus, accessibility of the brain parenchyma to 8-18C5 antibody was not sufficient to induce local demyelination. Therefore, it appears that activated encephalitogenic T cells are involved in initiating the 8-18C5 antibody-mediated demyelinating process.

Abstract

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.

Abstract

We used a new version of experimental autoimmune encephalomyelitis (EAE) in the rat to investigate immunotherapy of demyelination during autoimmune disease of the central nervous system (CNS). Encephalitis was induced by immunization of rats with myelin basic protein (MBP), and demyelination by systemic injection of a monoclonal antibody, 8-18C5, specific for a myelin/oligodendrocyte glycoprotein (MOG). Antibody injection resulted in hyperacute disease progression and extensive demyelination throughout the CNS. Immunotherapy of antibody-induced demyelination was possible with another monoclonal antibody, pta-3, specific for activated rat T cells. These findings demonstrate the synergy of T cell-mediated and antibody-dependent processes in rat CNS demyelination in vivo. Histologically, immunotherapy reduced the numbers of meningeal mononuclear cell inflammatory foci, but not parenchymal inflammation in the early phase of demyelinating disease. Animals which had received pta-3 antibody had less inflammation than untreated rats in the convalescent phase. Multiple pta-3 treatments most effectively suppressed inflammation. Furthermore, antibody-treated rats with demyelination developed a series of neurologic signs, including pronounced spasticity; that were not observed in control EAE rats and thus appears to be associated with the demyelinating process.

Abstract

Cellular expression and extracellular deposition of fibronectin and fibrin/fibrinogen in experimental allergic encephalomyelitis (EAE) in guinea pigs (GP) were studied by light and electron microscopic immunohistochemistry of the central nervous system (CNS). Normal GPs had few fibronectin+ and fibrin/fibrinogen+ CNS vessels. Positively stained vessels were more numberous in sensitized GPs prior to and after onset of EAE, and there were extracellular deposits of these molecules in inflammatory foci. Staining was greater in EAE-susceptible strain 13 GPs than in sensitized EAE-resistant strain 2 GP. On ultrastructural analysis both molecules were localized on endothelial cell luminal surfaces and on intravascular mononuclear cells. Fibronectin and the macrophage fibronectin receptor (A6F10) were found on mononuclear cells in parenchyma. Endothelial cell luminal surface fibronectin and fibrin/fibrinogen may mark and/or contribute to early immune events in EAE, including mononuclear cell margination and emigration in inflammatory sites. Genetic factors, cytokines, and components of coagulation may regulate these interactions in vivo.

Abstract

To analyze expression of the 2H4 (CD45R) Ag on inflammatory cells in the central nervous system immune response, immunohistochemical staining with a panel of anti-T cell mAb was performed on central nervous system tissues from 12 patients with multiple sclerosis (MS) and 8 patients with viral encephalitis. Only rare cells were stained with anti-2H4 in MS plaques, plaque edges, and adjacent white matter, whereas 2H4+ cells were more numerous in viral encephalitis (p less than 0.001). By contrast, no quantitative differences were found between MS plaque edges and viral encephalitis with anti-4B4 (helper-inducer function associated), anti-CD4, anti-CD3, and anti-IL-2R mAb, although there were fewer CD8+ cells in MS (p less than 0.01). These data indicate that the 2H4 Ag is selectively decreased and, because it is associated with suppressor-inducer function of CD4+ cells, that there may be a defect in the down-regulation of the in situ immune response in MS.

Abstract

Strains of mice with diverse genetic backgrounds were tested for susceptibility to experimental allergic encephalomyelitis (EAE) induced by myelin proteolipid protein. EAE was elicited in all strains of mice tested, but the clinical and histologic features varied. SJL (H-2s) mice had a high incidence of both clinical and histologic disease characterized by early onset of clinical signs. Inguinal lymph node T cells from diseased animals responded specifically [( 3H]thymidine incorporation) to proteolipid protein and not to myelin basic protein. In contrast, BALB/c (H-2d), DBA/1 (H-2q), C57BL/6 (H-2b), AKR (H-2k), CBA (H-2k), C3H (H-2k), B10.BR (H-2k), and C57BR (H-2k) mice showed a later onset of clinical signs and typically a lower disease incidence. However, the most marked variations in disease incidence occurred among BALB/c (H-2d) substrains in which the incidence of EAE ranged from eight of nine (BALB/cPt) to complete resistance (BALB/cWt and BALB/cORNL). Because these BALB/c substrains were initially derived from the same inbred genetic source and are serologically identical at H-2, these results suggest that expression of proteolipid protein-induced EAE in the mouse involves additional loci outside the MHC.

Abstract

To determine factors affecting major histocompatibility complex (MHC) molecule expression in situ in the human central nervous system (CNS) cryostat tissue sections from 36 autopsies and four biopsies were stained by immunoperoxidase with antibodies to class I (HLA-alpha chain, beta-2 microglobulin), class II (HLA-DR, HLA-DQ) MHC, lymphocyte, and macrophage antigens and Factor VIII-related antigen (VIII-RA). Stained cells and vessels/mm2 were counted in gray and white matter of four CNS anatomic levels. Class I molecules were found on parenchymal and endothelial cells (approximately 50% of VIII-RA + vessels) but not neurons, and were more abundant in gray than white matter (p less than 0.02). Class I molecules were absent in infants, but in adults expression was unaffected by age, sex, postmortem interval, presence of CNS lesions, or systemic illnesses. Expression of HLA-alpha chain and beta-2 microglobulin were the same. Class II molecules were usually absent but were found on parenchymal and endothelial cells in older adults, most frequently in association with macrophage infiltrates and spinal cord tract degenerations, but not with systemic illnesses. Expression of HLA-DR was greater than that of HLA-DQ. In the human CNS, regulation regulation of expression of MHC molecules is complex, can be affected by age, regional anatomy, and by local or remote CNS lesions, and may influence patterns and degrees of T cell immune responses.

Abstract

The factors contributing to chronic relapsing inflammatory disease processes of the central nervous system (CNS) and demyelination are poorly understood. In addition to cellular immune reactions, humoral factors such as antibodies might quantitatively or qualitatively influence the disease process. We therefore investigated the effects of administration of a monoclonal antibody specific for a CNS autoantigen on both acute and chronic experimental autoimmune encephalomyelitis (EAE) in mice and rats. This monoclonal antibody, 8-18C5, specific for a myelin/oligodendrocyte glycoprotein, was observed to accelerate clinical and pathologic changes of CNS autoimmune disease. In SJL mice with chronic relapsing EAE, injection of antibody into animals recovering from an attack induced fatal relapses; in Lewis rats, acute EAE was enhanced and associated with a hyperacute inflammatory response with demyelination, a feature not commonly seen in acute EAE. The demonstration that relapses and demyelination can be induced by administration of a white matter-reactive monoclonal antibody offers new possibilities to study processes resulting in CNS damage during autoimmune disease. Furthermore, these findings support the immunopathogenic potential of antibody to myelin components in inflammatory CNS disease processes and, specifically, in causing demyelination.

Abstract

Cell surface expression of Class II major histocompatibility complex (Ia) molecules is required for antigen recognition by T cells. To determine the ultrastructural cellular distribution of Ia molecules in the autoimmune disease model acute experimental allergic encephalomyelitis (EAE) we studied central nervous system (CNS) tissues from adult Strain 13 guinea pigs (GP). Experimental allergic encephalomyelitis was induced by sensitization with GP spinal cord homogenate in complete Freund's adjuvant (CFA). Nine of 11 sensitized GP had clinical and histologic EAE whereas unsensitized and CFA-sensitized controls were normal. Central nervous system tissues were reacted with monoclonal antibodies to either GP Ia or T cell surface antigen using an avidin-biotin immunoperoxidase technique and studied by electron microscopy; Ia was found on luminal but not abluminal surfaces of many meningeal and parenchymal vascular endothelial cells in GP with EAE. In EAE perivascular lymphocytes and macrophages and processes of unidentified cells in the parenchyma expressed surface Ia and Ia+ macrophages encircled and phagocytosed myelin. T cells were found predominantly in perivascular inflammatory cuffs. These observations indicate that following immunologic challenge Ia is expressed on luminal surfaces of vascular endothelium and on resident CNS cells, suggesting the possibility that these cells may have active antigen-presenting functions in CNS inflammatory reactions.

Abstract

To determine the effects of anti-T cell monoclonal antibody-induced systemic T cell depletion in neuro-autoimmune disease, we studied the in vivo effects of 8BE6, a mouse anti-guinea pig (GP) pan-T cell monoclonal antibody, on the course and immunopathology of the disease model experimental allergic encephalomyelitis (EAE) in adult Strain 13 GP. Central nervous system (CNS) tissues were studied by routine histology and by an immunoperoxidase staining technique using monoclonal antibodies to T cells, IgM, and macrophages. From 3 days before to 10 days after sensitization with GP spinal cord and complete Freund's adjuvant, the GP were given one or two i.p. doses of 3.4 mg 8BE6 or MOPC 21, the parent mouse myeloma ascites, or normal saline. Eighteen of 18 control-treated GP developed typical acute, paralytic EAE 11 to 21 days after sensitization, whereas acute EAE was prevented in 33 of 49 8BE6-treated GP (67%), and the onset was delayed and disease progression was slowed in the others. Five GP treated with 8BE6 from days 11 to 14 after sensitization, at the onset of neurologic signs, rapidly deteriorated within hours after treatment and had loss of T cell staining, and lymphocytolysis in the CNS. 8BE6-treated GP which did not develop acute EAE were observed daily for up to 700 days (mean = 213 days). Twenty-nine of 39 (74%) had from one to six relapses or fixed neurologic deficits. GP in relapse were additionally treated with 8BE6 (22), MOPC-21 (5), or saline (6) in a cross-over protocol. Clinical scores were improved from days 2 to 12 after treatment (p less than 0.05), and complete recovery within 30 days occurred more frequently (p = 0.046) and more rapidly (p less than 0.01), after 8BE6 as compared with control treatments. Recoveries occurred more often if 8BE6 was given early in the relapse. Multiple treatments led to dose-dependent levels of serum antibodies to mouse immunoglobulin detected by an ELISA. There were no differences between acute and chronic EAE in numbers of inflammatory foci or numbers of macrophages and T cells in CNS infiltrates, but GP with chronic EAE had more extensive demyelination and vascular fibrosis and more numerous IgM+ B cells in parenchymal and meningeal infiltrates than in acute EAE (p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract

As part of a study of the therapeutic potential of anti-T cell monoclonal antibodies, we studied the biologic effects of 8BE6, a mouse anti-guinea pig (GP) pan-T cell monoclonal antibody, on blood and tissue T cells and on the prototypic T cell-mediated reactions, classic delayed hypersensitivity (DH) and cutaneous basophil hypersensitivity (CBH). 8BE6 reacts to a 68,000 m.w. protein probably homologous with human CD5 (T1) and murine Lyt-1. A single dose of 1.8 to 3.4 mg 8BE6 caused lymphopenia and greater than 90% depletion of 8BE6+ peripheral T cells 1 to 72 hr later, and a significant but lesser decrease of lymphocytes reacting with another pan-T cell monoclonal antibody (p less than 0.02 at 24 hr). Free serum 8BE6 was detected for up to 48 hr after administration. Immunoperoxidase stains of tissue revealed that lymphocytes in lymph nodes and spleen were coated with mouse immunoglobulin 1 hr after antibody treatment and displayed in situ capping. Subsequently, there was a loss of T cells in all tissues (spleen, lymph node, liver, and kidney) except the thymus, with normal 8BE6 antigen staining returning by 72 hr. Areas of induration of DH reactions to PPD were reduced in 8BE6-treated GP, compared with pretreatment reactions in the same GP or in control-treated GP (p less than 0.001 for both). The numbers of infiltrating T cells and fibronectin-receptor-positive macrophages were also reduced. In contrast, 8BE6 had no effect on CBH reactions, as judged by erythema and basophil counts in 1-micron sections, although fewer T cells were found in reaction sites. There were no differences in IgM, fibronectin, or Ia staining between 8BE6-treated GP and controls. In vivo administration of a single dose of anti-T cell monoclonal antibody results in a transient, highly specific depletion of T cell populations in peripheral blood and tissues except the thymus. This treatment inhibits DH but not CBH reactions by systemic and local depletion of T cells.

Abstract

We studied in mice the in vivo pharmacokinetics and toxicity of murine monoclonal antibodies (MCA) and of disulfide-linked MCA conjugates of gelonin, a ribosomal inhibitor prepared from the seeds of Gelonium multiflorum. Iodinated MCA with specificity for human determinants and of gamma 1 or gamma 2a isotype had a circulatory half life (T 1/2) in the mouse of 4 days, which is consistent with previously published estimates of the circulatory T 1/2 of heterogeneous murine IgG. Iodinated murine MCA with specificity for murine determinants had a much shorter T 1/2, probably reflecting antigen binding. This effect could be partially overcome by the simultaneous injection of unlabeled MCA of identical specificity. Clearance of MCA-gelonin conjugates was characterized by an initial rapid phase lasting 8-12 h with a T 1/2 or from 4 to 7 h, followed by a slower clearance phase with T 1/2 approaching that of MCA. Moreover, the presence of significant amounts of intact conjugate in the murine circulation was demonstrable, by SDS gel electrophoresis, for up to 48 h post injection. Intraperitoneal injection of MCA-gelonin conjugate resulted in circulating levels identical to those achieved after i.v. administration after an initial 4 h equilibration. The LD50 of MCA-gelonin conjugates was approximately 25 mg/kg (i.v.) while that of gelonin was approximately 75 mg/kg (i.v.) MCA alone showed no toxicity in doses in excess of 150 mg/kg. At doses below the LD50 immunoconjugates caused a dose-dependent reversible weight loss. The main site of toxicity of MCA-gelonin conjugates was the liver; histopathological examination revealed dose-dependent foci of necrosis and acute inflammation. No pathology was observed in lung, spleen, kidney, gut or brain. The relationship to previous work in this area is discussed.

Abstract

To characterize the in situ cellular immune response in acute herpes simplex encephalitis (HSE), the authors stained frozen brain specimens from 19 patients with HSE and 8 controls with a panel of monoclonal antibodies, which allowed them to identify inflammatory cell subsets and expression of activation markers and major histocompatibility complex (MHC) molecules. Parenchymal and meningeal inflammatory infiltrates were composed of T cells, with fewer natural killer and B cells, and variable numbers of granulocytes and macrophages. T4+ and T8+ cells were present in equal numbers. Inflammatory cells expressed interleukin-2 receptor, MHC Class I (HLA-alpha chain, beta-2 microglobulin), and MHC Class II (HLA-DR, HLA-DQ) molecules. There was increased MHC molecule expression in HSE on resident cells, including neurons and vascular endothelium, and vascular expression of HLA-DR was greater than that of HLA-DQ (P less than 0.01). No correlations between immune parameters with amount of corticosteroid therapy or duration of illness before biopsy was performed or outcome were found. T cell-mediated cytotoxic and delayed hypersensitivity mechanisms may contribute to tissue injury in HSE.

Abstract

This study was undertaken to test the hypothesis that preferential responder strain-specific Ia expression can be detected in delayed hypersensitivity (DH) skin reactions. Seven adult (strain 2 X strain 13)F1 and two strain 13 guinea pigs were sensitized with poly-L glutamic acid-lysine (GL), poly-L glutamic acid-tyrosine (GT), and bovine insulin in complete Freund's adjuvant, and were skin tested with GL, GT, PPD, bovine insulin, porcine insulin (which has the same B chain as bovine insulin), and saline. Strain 2 guinea pigs react with bovine insulin A chain, GL, and PPD but not with GT or the bovine insulin B chain, whereas strain 13 guinea pigs react with bovine insulin B chain, GT, and PPD but not with GL or bovine insulin A chain. The (2 X 13)F1 animals had positive DH responses to GT, GL, PPD, and bovine insulin. At 24 hr, areas of induration were measured and the test sites and draining lymph nodes were biopsied. Cryostat sections were stained with monoclonal antibodies to strain 2 Ia, strain 13 Ia, and Ia framework determinants with immunoperoxidase. Stained dermal and subdermal inflammatory cells and vessels were counted on coded slides. In GT tests, there was more staining of dermal and subdermal cells and vessels for strain 13 Ia than strain 2 Ia (p less than 0.02). In bovine insulin tests there was more staining of dermal cells and vessels for strain 13 than strain 2 Ia (p less than 0.05). In GL tests there was more staining on dermal vessels and subdermal cells and vessels of strain 2 Ia than strain 13 Ia (p less than 0.05). There was much greater staining of strain 2 Ia of dermal cells and vessels in GL tests compared with strain 2 Ia staining in GT and bovine insulin tests (p less than 0.02, cells; p less than 0.01, vessels). No significant differences between strain 2 and strain 13 Ia expression were found in PPD, porcine insulin tests, saline controls, or in lymph nodes that drained sensitization sites from animals in which GL and GT had been injected on different sides. Anti-Ia framework expression generally correlated with the greater parental strain Ia in each reaction. These findings and previous observations in experimental allergic encephalomyelitis suggest that responder type Ia may be selectively found in vivo on mononuclear and endothelial cells in sites of T cell-mediated hypersensitivity reactions.

Abstract

Human T-cell lymphotropic virus type III (HTLV-III) has been isolated from neural tissues and cerebrospinal fluid (CSF) of patients with neurological syndromes associated with the acquired immune deficiency syndrome (AIDS) and the virus may be directly involved in the pathogenesis of the syndromes. To detect HTLV-III antigen in neural tissues from patients with AIDS, immunoperoxidase studies using a goat anti-HTLV-III serum were performed on frozen tissue sections of brain, spinal cord, and nerve from 13 patients with AIDS or HTLV-III-related neurological syndromes. HTLV-III was cultured from neural tissues or CSF in 11 of 13 of these patients. HTLV-III antigen was detected in the brains of 5 patients with AIDS and in none of the 13 non-AIDS control subjects. Rare positively stained cells were seen, frequently associated with capillaries and often located near microglial nodules. Morphologically, the cells resembled monocyte/macrophages and were found most frequently in the cortex of the frontal, temporal, and parietal lobes. These results provide further evidence that the subacute encephalitis of AIDS is associated with central nervous system infection by HTLV-III and that monocyte/macrophages are among the infected cell populations.

Abstract

A 42-year-old homosexual man without evidence of immune deficiency developed cerebral granulomatous angiitis in association with the isolation of human T-lymphotropic virus type III (HTLV-III) from brain tissue and cerebrospinal fluid. This syndrome may be an additional neurological sequela of HTLV-III infection.

Abstract

A chronic form of experimental allergic encephalomyelitis can be produced by sensitization of rabbits with bovine myelin proteolipid apoprotein (PLP). To investigate the humoral immune response in this model, serum PLP antibodies were determined by enzyme-linked immunosorbent and dot immunobinding assays. In an initial experiment, 3 PLP-sensitized rabbits with severe chronic experimental allergic encephalomyelitis had a positive antibody response whereas 3 with mild disease, or with no visible clinical disease, had no detectable antibodies against PLP. In a second experiment, 3 rabbits were preimmunized with PLP in incomplete Freund's adjuvant, followed by a single immunization with PLP in complete Freund's adjuvant. These animals developed chronic experimental allergic encephalomyelitis with different progression rates, although all eventually became severely paralyzed. In both experiments the anti-PLP response was maximal before or immediately after disease onset and tended to decline during disease progression. The degree of the anti-PLP response correlated with clinical and histologic disease severity. These data suggest a possible role for humoral factors in the modulation of the chronic EAE induced in PLP-immunized rabbits.

Abstract

The role of myelin proteolipid apoprotein (PLP) in the central nervous system (CNS) immune response of rabbits has been investigated by analyzing the immunopathology of chronic experimental allergic encephalomyelitis (EAE) induced by sensitization with PLP. Clinical disease occurred in seven out of nine rabbits sensitized with bovine PLP and monitored for up to 6 mo. Positive delayed hypersensitivity skin test reactions to PLP occurred in all but one of the PLP-sensitized animals. All PLP-sensitized animals had meningeal and CNS parenchymal inflammation that correlated with disease severity. Serial blood samples were stained with a panel of antibodies to rabbit T and B cells, as well as Ia, and large and small mononuclear cell populations were analyzed by flow cytometry. Peripheral leukocyte population staining did not correlate with clinical signs or sensitization to PLP. Cryostat CNS tissue sections were stained with the same set of antibodies by using an immunoperoxidase technique, and positive cells and vessels were counted. T cells and macrophages were numerous and in equal numbers in perivascular parenchymal inflammatory infiltrates, whereas B cells were less numerous (p less than 0.001). T cells also diffusely infiltrated the parenchyma. Most perivascular inflammatory cells and many scattered parenchymal cells were Ia+; Ia vascular expression was increased over controls (p less than 0.001), and also correlated with disease severity. The immunopathology of this chronic EAE model is the same as that of whole CNS tissue- and myelin basic protein-induced EAE in other species, and is similar to that of multiple sclerosis. Cellular immune responses to PLP may therefore contribute to systemic and in situ responses in CNS tissue demyelinating diseases.

Abstract

We report a microcystic meningioma of the falx cerebri which had an unusual histological pattern. It contained numerous palisaded structures in loose areas, giving the appearance of the loose and dense areas of a schwannoma. There were minimal meningeal whorls. Ultrastructural study confirmed the totally meningiomatous nature of this tumor.

Abstract

This study was undertaken to determine if there is differential expression of the relevant strain-specific Ia antigen on endothelial and parenchymal inflammatory cells in the central nervous systems (CNS) of guinea pigs (GP) with acute experimental allergic encephalomyelitis (EAE). Adult inbred GP were sensitized with GP spinal cord homogenate and complete Freund's adjuvant. Strain 13 GP and (2 x 13)F1 hybrids developed clinical disease within 2 to 3 wk after sensitization, whereas strain 2 GP did not, although all sensitized GP had CNS mononuclear inflammatory infiltrates. By using monoclonal antibodies to strain-specific and framework Ia epitopes with an immunoperoxidase technique, the distribution and amount of the strain 2 and strain 13 Ia were analyzed. Equivalent strain 2 and strain 13 Ia expression was found in normal tissues from F1 animals. Strain 2 and strain 13 GP sensitized for EAE had increased strain-specific Ia staining of CNS vessels and inflammatory cells over controls. However, F1 GP with EAE had markedly increased strain 13, but not strain 2, Ia on CNS parenchymal vessels and mononuclear inflammatory cells (p less than 0.001, for both). These results suggest for the first time that specific major histocompatibility complex gene products are selectively expressed on endothelial and inflammatory cells in situ in immune reactions in the target organ of individuals of heterogeneous immunogenetic composition.

Abstract

Acute experimental allergic encephalomyelitis (EAE) is a T cell-mediated, neurologic disease that is under immunogenetic control. We systematically analyzed the quantity and distribution of T cells, B cells, and macrophages in the central nervous system (CNS) of susceptible and resistant guinea (GP) with a panel of seven monoclonal antibodies by using the avidin-biotin complex (ABC) immunoperoxidase technique and alpha-naphthyl-butyrate esterase (ANBE) staining. Adult EAE-susceptible strain 13 GP immunized with isogeneic spinal cord homogenate (SC) or with myelin basic protein (MBP) developed clinical signs (paralysis, weight loss, etc.) in 2 to 3 wk. T cells were present in all CNS inflammatory foci and comprised 44% of the perivascular mononuclear cells. T cells diffusely infiltrated the neuropil away from inflammatory cell aggregates. These T cells were judged to be extravascular by the lack of an associated identifiable vessel in counter-stained sections, and by their persistence following exhaustive perfusion of the brains. In routine sections, mononuclear cells could be detected only in perivascular aggregates. IgM+ B cells comprised 9% of the perivascular infiltrates and did not diffusely infiltrate the parenchyma. ANBE+ macrophages comprised the remaining 47% of the identified perivascular cells. SC- and MBP-immunized GP showed equivalent numbers of inflammatory foci, T cells, and macrophages, but SC-immunized GP had more IgM+ cells in the meninges and choroid plexus (p less than 0.001, p less than 0.02, respectively). Virtually all cells in perivascular locations were Ia+. Ia+ mononuclear cells were also present in the neuropil. EAE-resistant strain 2 GP immunized with SC developed no clinical signs. These GP had fewer perivascular foci than strain 13 GP but, when present, the cellular composition, including the density of diffuse parenchymal T cell infiltrates, was indistinguishable. Significantly fewer parenchymal mononuclear cells in the strain 2 GP, however, displayed Ia, both in perivascular and diffuse infiltrates (p less than 0.001). We conclude that T cell migration into the CNS parenchyma is a characteristic feature of acute EAE in the GP, but that T cells can occur in this pattern without clinical signs of disease. The two features that distinguish susceptible and resistant strains were the frequency of perivascular infiltrates and the expression of Ia on parenchymal mononuclear cells, which probably reflects their enhanced immunologic activation in situ.

Abstract

The clinical and pathologic findings in 32 patients with central nervous system (CNS) coccidioidomycosis were studied. Seventeen patients had received more than 1.5 g of amphotericin B (AMB), chiefly intravenously, during treatment periods of up to eight years. Eight patients had received 246 mg to 1.3 g of AMB, and three patients had received only brief treatment (one to three days; total dose, no more than 100 mg). Fifteen patients had not received AMB. Significant clinical differences between the patients treated with and without AMB were longer survival time following diagnosis of illness (P less than 0.05) and more frequent cranial nerve signs in the treated patients (P = 0.089). The wide spectrum of macroscopic and microscopic lesions in the CNS included meningitis, ventriculitis, hydrocephalus, and cerebritis. Long-standing infections were associated with disseminated discrete foci of gliosis and infarcts in the brain, particularly in the basal ganglia and deep white matter, related to endarteritis obliterans in basilar meninges. In contrast to patients with CNS and systemic mycoses treated with amphotericin B methyl ester (J Infect Dis 146:125, 1982), no diffuse lesions of white matter were found in patients treated with or without AMB. Histopathologic patterns observed in this study included leptomeningitis alone, leptomeningitis with cerebritis, leptomeningitis with cerebritis and infarcts, and the unusual pattern of disseminated miliary granulomas. The frequency and extent of CNS lesions in the groups treated with and without AMB were not significantly different. It is concluded that AMB therapy, while prolonging survival, does not alter the spectrum of pathologic findings in CNS coccidioidomycosis infection.

Abstract

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated neuroimmunologic disease model characterized by meningeal and parenchymal mononuclear cell infiltrates (see preceding companion paper). Here we report enhanced staining for Ia in the central nervous system (CNS) microvasculature endothelium in acute EAE in adult strain 13 guinea pigs (GP) sensitized with GP spinal cord homogenate (SC) or with GP myelin basic protein (MBP) in complete Freund's adjuvant (CFA). Cryostat sections of CNS and other tissues were stained with two monoclonal antibodies, 5S2 and 22C4, to GP Ia determinants, and with polyclonal antibody to factor VIII-related antigen (VIII-RA) as an endothelial cell marker. Morphometric techniques were employed on immunoperoxidase counterstained and coded sections to determine the frequency of Ia+ vessels and cells. Rare (approximately 10% of VIII-RA+) vascular endothelial cells were Ia+ in the CNS of normal and CFA-sensitized controls. SC- or MBP-sensitized strain 13 GP sacrificed on day 7, before the onset of neurologic signs (pre-clinical), had no detectable CNS mononuclear cell infiltrates, but had increased (approximately 30% of VIII-RA+) endothelial cell Ia staining over controls (p less than 0.001). The endothelial Ia staining persisted (approximately 35% of VIII-RA+) in vessels as the animals developed paralysis. There were no differences in endothelial cell Ia between SC- and MBP-induced disease. EAE-resistant strain 2 GP sensitized with SC/CFA had no neurologic signs, and had fewer inflammatory foci than strain 13 GP with EAE, but had similar numbers of Ia+ endothelial cells. No differences in endothelial cell Ia staining were found in non-CNS tissues among any GP groups. In EAE, increased endothelial cell Ia is a pre-inflammatory, target organ-specific alteration that persists during inflammation. The findings suggest that in vivo modulation of endothelial cell Ia may be part of the local immune response. Endothelial cells may play a significant role, in antigen presentation or in promoting T cell migration, in the in situ immune response in the CNS.

Abstract

Clinical and autopsy studies of 14 patients treated with amphotericin B methyl ester (AME) for focal, disseminated, and nervous system mycotic infections revealed a high incidence of progressive neurologic dysfunction (dementia, akinesia, mutism, hyperreflexia, and tremor) and diffuse white matter degeneration. All of seven patients who received greater than 9.8 g of AME intravenously developed severe neurologic and neuropathologic changes. Two of three patients given 5-7.2 g of AME developed less severe neurologic symptoms; all three had mild diffuse white matter gliosis. Four patients given less than 1.5 g of AME had no bran abnormalities except those related to coccidioidal meningitis. Thirty-one control patients who died on untreated or amphotericin B-treated coccidioidal meningitis showed no diffuse white matter abnormalities. These findings indicate that prolonged administration of AME and/or other contaminating polyenes injures human white matter. Long-term animal studies, with particular attention to nervous system histology, must precede human use of other polyene derivatives.

Abstract

A 28-year-old woman with bilateral headaches and vomiting was found to have normal prolactin levels despite an eight-year history of intermittent galactorrhea and amenorrhea and the current finding of a pituitary microadenoma. The microadenoma contained hemosiderin. It is concluded that pituitary apoplexy is not confined to large tumors that have outgrown their blood supply, but can occur in microadenomas with regression of a positive endocrinopathy.

Abstract

Basal ganglia, thalamus, cerebral cortex, and subcortical white matter were studied in ten cases of hepatic encephalopathy (HE), including three cases of acquired hepatocerebral degeneration (HCD), and in thirteen age-matched controls using the peroxidase-antiperoxidase immunohistochemical staining technique for glial fibrillary acidic (GFA) protein. HE cases all had pronounced Alzheimer type II astrocytosis. The perikarya and processes of Alzheimer type II glia did not stain for GFA protein. Staining of perivascular endfeet was evaluated by first selecting blood vessels throughout the gray and white matter in hematoxylin and eosin-stained slides to eliminate bias. The vessels were then identified in sections stained for GFA protein and graded as to complete circumferential, partial circumferential, or absence of staining. Both the degree and frequency of staining in the basal ganglia, thalamus, and cerebral cortex were significantly decreased in cases of HE; no statistically significant differences were found for the white matter. There were no significant differences in staining between HCD and other HE cases. These findings show that the Alzheimer II change is associated with a loss of immunohistochemically detectable GFA protein in cerebral gray matter.

Abstract

A young male with Buerger's disease who had previously required a left leg amputation died in renal failure and sepsis. Postmortem examination revealed an obliterative lesion of the celiac artery, which resulted in hepatic, splenic, and pancreatic infarctions. Celiac artery involvement represents an unusual manifestation of thromboangiitis obliterans.